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Methods in
Molecular Biology 2625
Sanjoy K. Bhattacharya
Editor
Lipidomics
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
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constitute the key ingredient in each and every volume of the Methods in Molecular Biology
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Lipidomics
Methods and Protocols
Second Edition
Edited by
Bascom Palmer Eye Institute, University of Miami, Miami, FL, USA
Editor
Bascom Palmer Eye Institute
University of Miami
Miami, FL, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
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Preface
The history of lipids spans almost four centuries. In 1673, Techenius Otto first suggested
the presence of lipids. He described acidic compounds in fat by noting that the alkali is
neutralized by animal fat in the process of soap manufacturing. Lipid identification and
quantification has come a long way since then. Mammalian cell boundaries are demarcated
by lipids. In addition to being boundary molecules, they are also highly efficient signaling
molecules. Each fragment of a lipid can potentially generate a different signal in living cells,
thus emphasizing the importance of lipid research from the perspectives of fundamental
biology and synthetic organic chemistry. However, lipids are not a dominant theme in most
biological laboratories despite a rich history of lipid research. A tenth or less of the
laboratories engaged in biomedical research focus on lipid biochemistry. This is partly due
to lack of easily available reagents and otherwise due to a lack of awareness regarding the
tools, techniques, and knowledge of protocols. For example, antibodies to most lipids are
neither available nor easily generated. However, in the last 5 years alone, tremendous
advancements have been made in lipid identification and quantification methods. This
includes major advancements in mass spectrometry and bioinformatics analyses. A search
with keyword “lipidomics” resulted in over 1000 entries in PUBMED for the first time in
2019 and has been growing ever since. “Proteomics” had already reached this level in 2001.
A wealth of clones of different enzymes and lipid-handling proteins have been accumulated
over several years. Generation of mouse models with specific alterations in lipid metabolizing
enzymes has paved the way for tremendous advancements in other techniques. This book
presents an account of areas of utility, techniques, and bioinformatic advancements. We
expect this volume of Methods in Molecular Biology to be useful to biochemists, molecular
biologists, neuroscientists, vision research scientists, as well as all biomedical researchers with
interest in disease discovery and drug development. The chapters here begin with protocols
for lipid isolation and extraction. Some isolation is specific for tissue, cell, or organelle. While
general lipid extraction procedures are more than 50 years old, specific tweaks are immensely
helpful. This collection for lipidomics moves from extractive mass spectrometry to imaging
mass spectrometry methods allowing localization of lipids in tissues, which is advancing and
likely to expand to cells or organelles in the near future. Some of these protocols are
expected to aid investigation of pathologic deposits and fluorescence in correct cellular
layers within the tissue. Several different high-throughput mass spectrometric approaches,
computational and function analysis of lipids, and database and bioinformatics analysis
methods are also included. These protocols have been complemented by methods addres-
sing specific problems from membranes, fractionated subcellular compartments, or orga-
nelles to whole organisms. In the future, these protocols will prove useful in the deeper and
integrative investigation of biological questions in biomedical research laboratories
worldwide.
Miami, FL, USA Sanjoy K. Bhattacharya
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Isolation of Mitochondrial Lipids and Mass Spectrometric Analysis . . . . . . . . . . . 1

Alexa M. Jauregui, Zoé M. Cubero Cortés, Sean D. Meehan,
and Sanjoy K. Bhattacharya
2 Isolation of Neuronal Synaptic Membranes by Sucrose
Gradient Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Blake R. Hopiavuori, Dustin R. Masser, Joseph L. Wilkerson,
Richard S. Brush, Nawajes A. Mandal, Robert E. Anderson,
and Willard M. Freeman
3 Multi-macromolecular Extraction from Endosymbiotic Anthozoans. . . . . . . . . . . 17
Anderson B. Mayfield
4 High-Resolution Liquid Chromatography–Mass Spectrometry
for Lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Faith Christine Harvey, Vanessa Collao, and Sanjoy K. Bhattacharya
5 Characterization of Gangliosides from Mouse Optic Nerve Samples
Using Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Jennifer Arcuri and Sanjoy K. Bhattacharya
6 Protocol and Methods Applicable to Retinal Vascular Diseases. . . . . . . . . . . . . . . . 71
Saurabh Kumar, Manjunath B. Joshi, and Inderjeet Kaur
7 Analysis and Annotation of Phospholipids by Mass
Spectrometry-Based Metabolomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Álvaro González-Domı́nguez, Marı́a Santos-Martı́n, Ana Sayago,
Alfonso Marı́a Lechuga-Sancho, Ángeles Fernández-Recamales,
and Raúl González-Domı́nguez
8 Profiling the Mammalian Lipidome by Quantitative Shotgun Lipidomics . . . . . . 89
Mads Møller Foged, Kenji Maeda, and Mesut Bilgin
9 Lipidomics Analysis with Triple Quadrupole Mass Spectrometry . . . . . . . . . . . . . . 103
Meher A. Saleem, Anna Mueller, and Sanjoy K. Bhattacharya
10 High-Performance Chromatographic Separation of Cerebrosides . . . . . . . . . . . . . 107
Renaud Sicard
11 Quantification of Endocannabinoids from Biological Samples
Using Liquid Chromatography–Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . 115
Zhiying Wang, Chenglin Mo, Lynda Bonewald, and Marco Brotto
12 Analysis of Lipids, Fatty Acid, and Cholesterol in Membrane
Microdomains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Martin-Paul Agbaga, Mark E. McClellan, and Michael H. Elliott
vii
viii Contents
13 Analysis of Cholesterol Lipids Using Gas Chromatography

Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Emily J. Neag, Vanessa Collao, and Sanjoy K. Bhattacharya
14 Analyses and Localization of Phosphatidylcholine and Phosphatidylserine
in Murine Ocular Tissue Using Imaging Mass Spectrometry . . . . . . . . . . . . . . . . . 149
Varun Krishnan, Sean D. Meehan, Colin Hayter,
15 A Robust Phenotypic Screening Assay Utilizing Human Podocytes
to Identify Agents that Modulate Lipid Droplets . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Margaret Gurumani, Shamroop Kumar Mallela, Javier Varona,
Sandra Merscher, Alessia Fornoni, and Hassan Al-Ali
16 Capillary Electrophoresis Plasma Fractionation for Lipids Analysis . . . . . . . . . . . . 175
Anddre Osmar Valdivia, Carolyn Cray, and Sanjoy K. Bhattacharya
17 Combined Use of MALDI-TOF Mass Spectrometry and
31
P NMR Spectroscopy for the Analysis of (Phospho)Lipids . . . . . . . . . . . . . . . . . 183
Jenny Leopold, Kathrin M. Engel, Patricia Prabutzki,
and Jürgen Schiller
18 Single-Step Capture and Targeted Metabolomics of Alkyl-Quinolones
in Outer Membrane Vesicles (OMVs) of Pseudomonas Aeruginosa . . . . . . . . . . . . 201
Pallavi Lahiri, Priyakshi Gogoi, and Dipankar Ghosh
19 Isoprenylation of Monomeric GTPases in Human Trabecular
Meshwork Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Evan B. Stubbs Jr
20 Angiogenesis Model of Cornea to Understand the Role of
Sphingosine 1-Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Joseph L. Wilkerson, Sandip K. Basu, and Nawajes A. Mandal
21 Lipid Extraction and Sample Preservation Techniques for Stable
Isotope Analysis and Ecological Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Kyle H. Elliott, James D. Roth, Kevin Crook, and David Yurkowski
22 Rapid Analysis of Plasmalogen Individual Species by High-Resolution
Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Glenda Vasku, Niyazi Acar, and Olivier Berdeaux
23 Untargeted Analysis of Lipids Containing Very Long Chain Fatty
Acids in Retina and Retinal Tight Junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Julia V. Busik, Hannah Decot, Anna B. Lin, and Todd A. Lydic
24 Analysis of Lipid Contents in Human Trabecular Meshwork Cells
by Multiple Reaction Monitoring (MRM) Profiling Lipidomics . . . . . . . . . . . . . . 291
Ting Wang and Padmanabhan Paranji Pattabiraman
25 Mass Spectrometry Approaches for Detection and Determination
of Prostaglandins from Biological Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Zhiying Wang, Chenglin Mo, Kamal Awad, Lynda Bonewald,
and Marco Brotto
26 Downloading and Analysis of Metabolomic and Lipidomic Data
from Metabolomics Workbench Using MetaboAnalyst 5.0 . . . . . . . . . . . . . . . . . . . 313
Ashley Howell and Charles Yaros
Contents ix
27 Accurate Analysis of Lipid Building Blocks Using the Tool LipidOne . . . . . . . . . 323
Roberto Maria Pellegrino, Matteo Giulietti, Husam B. R. Alabed,
Anna Aurora Taddei, Sandra Buratta, Lorena Urbanelli,
Francesco Piva, and Carla Emiliani
28 Image-Based Longitudinal Characterization of Corneal Wound
to Understand the Role of Sphingosine-1-Phosphate. . . . . . . . . . . . . . . . . . . . . . . . 337
Sandip K. Basu and Nawajes Mandal
29 Characterization of Trabecular Meshwork Mechanical Property
Modulation After Application of Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Wyndham Batchelor, Jennifer Arcuri, Sanjoy K. Bhattacharya,
and Noël M. Ziebarth
30 C-Laurdan: Membrane Order Visualization of HEK293t Cells
by Confocal Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Sean D. Meehan, Colin Hayter, and Sanjoy K. Bhattacharya
31 Sonication-Based Basic Protocol for Liposome Synthesis. . . . . . . . . . . . . . . . . . . . . 365
Roberto Mendez
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Contributors
NIYAZI ACAR • Eye and Nutrition Research Group, Centre des Sciences du Goût et de
l’Alimentation, CNRS, INRAE, Institut Agro, Université de Bourgogne Franche-Comté,
Dijon, France
MARTIN-PAUL AGBAGA • Department of Ophthalmology/Dean McGee Eye Institute,
University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; Department of
Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA;
Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center,
Oklahoma City, OK, USA
HUSAM B. R. ALABED • Department of Chemistry, Biology and Biotechnology, University of
Perugia, Perugia, Italy
HASSAN AL-ALI • Katz Family Division of Nephrology and Hypertension, Department of
Medicine, University of Miami Miller School of Medicine, Florida, USA; Peggy and Harold
Katz Family Drug Discovery Center, University of Miami Miller School of Medicine,
Florida, USA; Department of Neurological Surgery, University of Miami Miller School of
Medicine, Florida, USA; The Miami Project to Cure Paralysis, University of Miami Miller
School of Medicine, Florida, USA; Sylvester Comprehensive Cancer Center, University of
Miami Miller School of Medicine, Florida, USA
ROBERT E. ANDERSON • Oklahoma Center for Neuroscience, University of Oklahoma Health
Sciences Center, Oklahoma City, OK, USA; Department of Ophthalmology, Dean McGee
Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA;
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma
City, OK, USA
JENNIFER ARCURI • Bascom Palmer Eye Institute & Department of Ophthalmology,
University of Miami, Miami, FL, USA; Molecular and Cellular Pharmacology Graduate
Program, University of Miami, Miami, FL, USA; Miami Integrative Metabolomics
Research Center, University of Miami, Miami, FL, USA
KAMAL AWAD • Bone-Muscle Research Center, College of Nursing & Health Innovation,
University of Texas at Arlington, Arlington, TX, USA
SANDIP K. BASU • Department of Ophthalmology, University of Tennessee Health Sciences
Center, Memphis, TN, USA; Hamilton Eye Institute, Department of Ophthalmology,
University of Tennessee Health Science Center, Memphis, TN, USA
WYNDHAM BATCHELOR • Biomedical Atomic Force Microscopy Laboratory, Department of
Biomedical Engineering, University of Miami College of Engineering, Miami, FL, USA
OLIVIER BERDEAUX • Eye and Nutrition Research Group, Centre des Sciences du Goût et de
Dijon, France; PROBE Research Infrastructure, ChemoSens Facility, Dijon, France
SANJOY K. BHATTACHARYA • Bascom Palmer Eye Institute & Department of Ophthalmology,
University of Miami, Miami, FL, USA; Molecular and Cellular Pharmacology Graduate
Program, University of Miami, Miami, FL, USA; Miami Integrative Metabolomics
Research Center, University of Miami, Miami, FL, USA; Bascom Palmer Eye Institute,
University of Miami Miller School of Medicine, Miami, FL, USA; Bascom Palmer Eye
Institute, Miller School of Medicine at University of Miami, Miami, FL, USA; Miami
xi
xii Contributors
Integrative Metabolomics Research Center, Miami, FL, USA; Molecular Cellular

Pharmacology Graduate Program, University of Miami, Miami, FL, USA
MESUT BILGIN • Cell Death and Metabolism Unit, Center for Autophagy, Recycling and
Disease, Danish Cancer Society Research Center, Copenhagen, Denmark; Lipidomics Core
Facility, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research
Center , Copenhagen, Denmark
LYNDA BONEWALD • Indiana Center for Musculoskeletal Health, Indiana University Medical
School, Indianapolis, IN, USA; Indiana Center for Musculoskeletal Health, Indiana
University Medical School, Indianapolis, IN, USA
MARCO BROTTO • Bone-Muscle Research Center, College of Nursing & Health Innovation,
RICHARD S. BRUSH • Department of Ophthalmology, Dean McGee Eye Institute, University of
Oklahoma Health Sciences Center, Oklahoma City, OK, USA
SANDRA BURATTA • Department of Chemistry, Biology and Biotechnology, University of
JULIA V. BUSIK • Department of Physiology, Michigan State University, East Lansing, MI,
USA
VANESSA COLLAO • Department of Ophthalmology, Bascom Palmer Eye Institute, Miller
School of Medicine at University of Miami, Miami, FL, USA; Department of
Ophthalmology, Bascom Palmer Eye Institute, Miller School of Medicine at University of
Miami, Miami, FL, USA
CAROLYN CRAY • Division of Comparative Pathology, Department of Pathology & Laboratory
Medicine, University of Miami, Miami, FL, USA
KEVIN CROOK • Department of Biological Sciences, University of Manitoba, Winnipeg, MB,
Canada
ZOÉ M. CUBERO CORTÉS • San Juan Bautista School of Medicine, Caguas, PR, USA
HANNAH DECOT • Department of Physiology, Michigan State University, East Lansing, MI,
USA
KYLE H. ELLIOTT • Department of Natural Resource Sciences, McGill University, Montreal,
QC, Canada
MICHAEL H. ELLIOTT • Department of Ophthalmology/Dean McGee Eye Institute,
University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; Oklahoma
Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City,
OK, USA; Department of Physiology, University of Oklahoma Health Sciences Center,
Oklahoma City, OK, USA
CARLA EMILIANI • Department of Chemistry, Biology and Biotechnology, University of
KATHRIN M. ENGEL • Faculty of Medicine, Institute for Medical Physics and Biophysics,
Leipzig University, Leipzig, Germany
ÁNGELES FERNÁNDEZ-RECAMALES • Agrifood Laboratory, Faculty of Experimental Sciences,
University of Huelva, Huelva, Spain; International Campus of Excellence CeiA3,
University of Huelva, Huelva, Spain
MADS MØLLER FOGED • Cell Death and Metabolism Unit, Center for Autophagy, Recycling
and Disease, Danish Cancer Society Research Center, Copenhagen, Denmark
ALESSIA FORNONI • Katz Family Division of Nephrology and Hypertension, Department of
Florida, USA
Contributors xiii
WILLARD M. FREEMAN • Genes & Human Disease Program, Oklahoma Medical Research
Foundation, Oklahoma City, USA
DIPANKAR GHOSH • Special Center for Molecular Medicine, Jawaharlal Nehru University,
New Delhi, India
MATTEO GIULIETTI • Department of Specialistic Clinical and Odontostomatological Sciences,
Polytechnic University of Marche, Ancona, Italy
PRIYAKSHI GOGOI • Special Center for Molecular Medicine, Jawaharlal Nehru University,
New Delhi, India
ÁLVARO GONZÁLEZ-DOMÍNGUEZ • Instituto de Investigacion e Innovacion Biomédica de
Cádiz (INiBICA), Hospital Universitario Puerta del Mar, Universidad de Cádiz, Cádiz,
Spain
RAÚL GONZÁLEZ-DOMÍNGUEZ • Instituto de Investigacion e Innovacion Biomédica de Cádiz
(INiBICA), Hospital Universitario Puerta del Mar, Universidad de Cádiz, Cádiz, Spain
MARGARET GURUMANI • Katz Family Division of Nephrology and Hypertension, Department
of Medicine, University of Miami Miller School of Medicine, Florida, USA; Peggy and
Harold Katz Family Drug Discovery Center, University of Miami Miller School of
Medicine, Florida, USA
FAITH CHRISTINE HARVEY • Department of Ophthalmology, Bascom Palmer Eye Institute,
Miller School of Medicine at University of Miami, Miami, FL, USA; Miami Integrative
Metabolomics Research Center, Miami, FL, USA
COLIN HAYTER • Bascom Palmer Eye Institute, Miller School of Medicine at University of
Miami, Miami, FL, USA; Miami Integrative Metabolomics Research Center, Miami, FL,
USA; University of Miami Miller School of Medicine, Miami, FL, USA
BLAKE R. HOPIAVUORI • Oklahoma Center for Neuroscience, University of Oklahoma Health
Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
ASHLEY HOWELL • Department of Ophthalmology, Miller School of Medicine, University of
Miami, Miami, FL, USA
ALEXA M. JAUREGUI • Bascom Palmer Eye Institute, Miller School of Medicine at University of
MANJUNATH B. JOSHI • Department of Ageing Research, Manipal School of Life Sciences,
Manipal Academy of Higher Education, Manipal, Karnataka, India
INDERJEET KAUR • Prof Brien Holden Eye Research Centre, L V Prasad Eye Institute,
Hyderabad, Telangana, India
VARUN KRISHNAN • Bascom Palmer Eye Institute, Miller School of Medicine at University of
SAURABH KUMAR • Prof Brien Holden Eye Research Centre, L V Prasad Eye Institute,
Hyderabad, Telangana, India; Manipal Academy of Higer Education, Manipal, India
PALLAVI LAHIRI • Special Center for Molecular Medicine, Jawaharlal Nehru University, New
Delhi, India
ALFONSO MARÍA LECHUGA-SANCHO • Instituto de Investigacion e Innovacion Biomédica de
Cádiz (INiBICA), Hospital Universitario Puerta del Mar, Universidad de Cádiz, Cádiz,
Spain; Departamento Materno Infantil y Radiologı́a, Facultad de Medicina, Universidad
de Cádiz, Cádiz, Spain; Unidad de Endocrinologı́a Pediátrica y Diabetes, Servicio de
Pediatrı́a, Hospital Universitario Puerta del Mar, Cádiz, Spain
xiv Contributors
JENNY LEOPOLD • Faculty of Medicine, Institute for Medical Physics and Biophysics, Leipzig
University, Leipzig, Germany
ANNA B. LIN • Department of Physiology, Michigan State University, East Lansing, MI, USA
TODD A. LYDIC • Department of Physiology, Michigan State University, East Lansing, MI,
USA
KENJI MAEDA • Cell Death and Metabolism Unit, Center for Autophagy, Recycling and
Disease, Danish Cancer Society Research Center, Copenhagen, Denmark
SHAMROOP KUMAR MALLELA • Katz Family Division of Nephrology and Hypertension,
Department of Medicine, University of Miami Miller School of Medicine, Florida, USA;
Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School
of Medicine, Florida, USA
NAWAJES A. MANDAL • Oklahoma Center for Neuroscience, University of Oklahoma Health
Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA;
Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City,
OK, USA; Memphis VA Medical Center, Memphis, TN, USA; Department of
Ophthalmology, University of Tennessee Health Sciences Center, Memphis, TN, USA;
Departments of Anatomy and Neurobiology, and Pharmaceutical Sciences, University of
Tennessee Health Sciences Center, Memphis, TN, USA; Hamilton Eye Institute,
Department of Ophthalmology, University of Tennessee Health Science Center, Memphis,
TN, USA
DUSTIN R. MASSER • Department of Physiology, University of Oklahoma Health Sciences
Center, Oklahoma City, OK, USA; Reynolds Oklahoma Center on Aging, University of
Oklahoma Health Sciences Center, Oklahoma City, OK, USA
ANDERSON B. MAYFIELD • Coral Reef Diagnostics, Miami, United States
MARK E. MCCLELLAN • Department of Ophthalmology/Dean McGee Eye Institute,
University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
SEAN D. MEEHAN • Bascom Palmer Eye Institute, Miller School of Medicine at University of
ROBERTO MENDEZ • Department of Pediatrics, University of Wisconsin, Madison, WI, USA
SANDRA MERSCHER • Katz Family Division of Nephrology and Hypertension, Department of
Florida, USA
CHENGLIN MO • Bone-Muscle Research Center, College of Nursing & Health Innovation,
ANNA MUELLER • Bascom Palmer Eye Institute, University of Miami Miller School of
Medicine, Miami, FL, USA; Miami Integrative Metabolomics Research Center, Miami,
FL, USA
EMILY J. NEAG • Department of Ophthalmology, Bascom Palmer Eye Institute, Miller School
of Medicine at University of Miami, Miami, FL, USA; Miami Integrative Metabolomics
Research Center, Miami, FL, USA; Michigan State University College of Osteopathic
Medicine, East Lansing, MI, USA
PADMANABHAN PARANJI PATTABIRAMAN • Glick Eye Institute, Department of Ophthalmology,
Indiana University School of Medicine, Indianapolis, IN, USA; Stark Neuroscience
Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA
Contributors xv
ROBERTO MARIA PELLEGRINO • Department of Chemistry, Biology and Biotechnology,

University of Perugia, Perugia, Italy
FRANCESCO PIVA • Department of Specialistic Clinical and Odontostomatological Sciences,
Polytechnic University of Marche, Ancona, Italy
PATRICIA PRABUTZKI • Faculty of Medicine, Institute for Medical Physics and Biophysics,
Leipzig University, Leipzig, Germany
JAMES D. ROTH • Department of Biological Sciences, University of Manitoba, Winnipeg, MB,
Canada
MEHER A. SALEEM • Bascom Palmer Eye Institute, University of Miami Miller School of
Medicine, Miami, FL, USA; Miami Integrative Metabolomics Research Center, Miami,
FL, USA; Georgetown University School of Medicine, Washington, DC, USA
MARÍA SANTOS-MARTÍN • Agrifood Laboratory, Faculty of Experimental Sciences, University
of Huelva, Huelva, Spain; International Campus of Excellence CeiA3, University of
Huelva, Huelva, Spain
ANA SAYAGO • Agrifood Laboratory, Faculty of Experimental Sciences, University of Huelva,
Huelva, Spain; International Campus of Excellence CeiA3, University of Huelva, Huelva,
Spain
JÜRGEN SCHILLER • Faculty of Medicine, Institute for Medical Physics and Biophysics, Leipzig
University, Leipzig, Germany
RENAUD SICARD • Department of Biochemistry and Molecular Biology, Sylvester
Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL,
USA
EVAN B. STUBBS JR • Department of Veterans Affairs, Edward Hines Jr. VA Hospital, Hines,
IL, USA; Department of Ophthalmology, Stritch School of Medicine, Loyola University
Chicago, Maywood, IL, USA
ANNA AURORA TADDEI • Department of Chemistry, Biology and Biotechnology, University of
LORENA URBANELLI • Department of Chemistry, Biology and Biotechnology, University of
ANDDRE OSMAR VALDIVIA • University of Minnesota Medical School, Minneapolis, MN, USA
JAVIER VARONA • Katz Family Division of Nephrology and Hypertension, Department of
Florida, USA
GLENDA VASKU • Eye and Nutrition Research Group, Centre des Sciences du Goût et de
Dijon, France; PROBE Research Infrastructure, ChemoSens Facility, Dijon, France
TING WANG • Glick Eye Institute, Department of Ophthalmology, Indiana University School
of Medicine, Indianapolis, IN, USA; Stark Neuroscience Research Institute, Indiana
University School of Medicine, Indianapolis, IN, USA
ZHIYING WANG • Bone-Muscle Research Center, College of Nursing & Health Innovation,
JOSEPH L. WILKERSON • Department of Ophthalmology, Dean McGee Eye Institute,
University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; Department of
Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA;
Department of Nutrition and Integrative Physiology, University of Utah, Salt Lake City,
UT, USA
xvi Contributors
CHARLES YAROS • Department of Ophthalmology, University of Miami, McKnight Vision

Research Center, Miami, FL, USA
DAVID YURKOWSKI • Fisheries and Oceans Canada, Arctic and Aquatic Research Division,
Winnipeg, MB, Canada
NOËL M. ZIEBARTH • Biomedical Atomic Force Microscopy Laboratory, Department of
Biomedical Engineering, University of Miami College of Engineering, Miami, FL, USA
Chapter 1
Isolation of Mitochondrial Lipids and Mass Spectrometric

Analysis
Alexa M. Jauregui, Zoé M. Cubero Cortés, Sean D. Meehan,
Abstract
Mitochondria participate in many important metabolic processes in the body. The lipid profile of mito-
chondria is especially important in membrane regulation and pathway signaling. The isolation and study of
these lipids can provide unparalleled information about the mechanisms behind these cellular processes. In
this chapter, we describe a protocol to isolate mitochondrial lipids from homogenized murine optic nerves.
The lipid extraction was performed using butanol–methanol (BUME) and subsequently analyzed using
liquid chromatography–mass spectrometry. Further analysis of the raw data was conducted using Lipid-
Search™ and MetaboAnalyst 4.0.
Key words Mitochondrial lipids, Neurodegeneration, Lipidomics, Liquid chromatography, mass

spectrometry
1 Introduction
Mitochondria are gaining rapid popularity among translational

researchers due to their central metabolic function and ability to
affect many organ systems. Mitochondrial dysfunction is involved
in the pathophysiology of several diseases and is indicative of neu-
rodegenerative disorders [1–3]. Mitochondria are elongated bands
or tube-shaped organelles with an inner membrane folded into
structures known as cristae. Understanding the shape and function
of cristae can give us insight into how the organelle’s morphology
affects neurodegeneration.
For instance, mitochondrial cristae contain enzymes of the
electron transport chain and ATP synthase. In turn, dysfunction
and shifts in the organelle can alter oxidative phosphorylation and
respiratory activity [4].
Mitochondrial membranes are composed of several types of
phospholipids that are responsible for its morphology. The inner
Sanjoy K. Bhattacharya (ed.), Lipidomics: Methods and Protocols,

Methods in Molecular Biology, vol. 2625, https://doi.org/10.1007/978-1-0716-2966-6_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023
1
2 Alexa M. Jauregui et al.
mitochondrial membrane (IMM) contains approximately 20% of

cardiolipin (CL), a specific phospholipid found in the IMM [5]. CL
is responsible for efficient oxidative phosphorylation and is an
important structural membrane component [1, 5]. Studying mito-
chondrial lipids like cardiolipin provide insight on the role lipids
play in complex metabolic pathways and how they can lead to both
dysregulation and disease. Understanding the structure, function,
and lipid composition of the mitochondria can help researchers
identify aberrant lipid shifts to develop therapeutic targets for
mitochondrial disorders [4].
Given the importance of purity for lipidomics analyses, ultra-
centrifugation (UC) is a mitochondrial isolation technique that has
shown high enrichment in mitochondria-specific cardiolipins as
well as their precursors, in comparison to other isolation methods.
UC has been shown to yield a pure mitochondrial fraction with
little contamination by non-mitochondrial organelles (e.g., endo-
plasmic reticulum). While isolation of mitochondria by ultracentri-
fugation (UC) is recommended [6], differential centrifugation
(DC) can also be used due to its simplicity, cost, and ability to
obtain large amounts of mitochondria in a short period of time [7].
In this chapter, we demonstrate the use of a modified butanol–
methanol (BUME) lipid extraction [8, 9]. We opted for the BUME
method instead of the gold standard Bligh and Dyer method [10]
after determining that the former method extracted more lipid
species of higher grade in our small sample size (~1 mg). This
chapter gives a protocol for mitochondrial lipid extraction using
differential centrifugation with BUME in murine optic nerve. A Q
Exactive Orbitrap Mass Spectrometer was used to generate the raw
data for each sample. All raw data was uploaded and processed with
LipidSearch™ and normalized and exported for bioinformatics
analysis using MetaboAnalyst 4.0.
2 Materials
Prepare all solutions using LC-MS grade solvents and chemicals to

ensure samples are extracted using solvents of the utmost purity.
2.1 Solvents/ 1. Mitochondria Isolation Kit for Tissue (PIERCE, Rockford, IL,
Solutions USA, Cat. No. 89801) (see Note 1).
2. 1× phosphate-buffered saline (PBS).
3. BupH™ phosphate-buffered saline (BupH PBS).
4. Wash buffer.
5. N-butanol/methanol 3:1 (v/v).
6. Heptane/ethyl acetate 3:1 (v/v).
7. 50 mM lithium chloride.
Mass Spectrometry Analysis of Isolated Mitochondrial Lipids 3
8. Pierce Micro BCA Protein Assay Kit.

9. Chloroform/methanol 2:1 (v/v).
10. Mobile Phase A: methanol/water 60:40 (v/v) with 10 mM
ammonium acetate.
11. Mobile Phase B: methanol/chloroform 60:40 (v/v) with
10 mM ammonium acetate.
2.2 Other Materials 1. Dounce homogenizer.

2. Centrifuge.
3. Vortexer.
4. Ice.
5. Disposable Borosilicate Glass Pasteur Pipet.
6. SpeedVac Concentrator.
7. Ultrasonic water bath.
8. Pierce Micro BCA Protein Assay Kit.
2.3 High- 1. Accucore™ Vanquish™ C18+ Column

Performance Liquid (150 mm × 2.1 mm × 1.5 μm, Thermo Scientific).
Chromatography and 2. Q Exactive Orbitrap Mass Spectrometer (Thermo Scientific).
Mass Spectrometry
3. Heated electrospray ionization source (HESI).
4. Thermo Fisher™ LipidSearch™ software.
3 Methods
3.1 Sample 1. Dry and weigh optic nerves to approximately 1 mg.

Preparation and Wash optic nerves twice in 1× phosphate-buffered saline
Mitochondrial (PBS) (see Note 2).
Isolation 2. Resuspend samples in 200 μL of BupH PBS.
3. Using a dounce homogenizer, carefully dounce the optic nerve
tissue 15 times.
4. Centrifuge homogenate at 1000 × g at 4 °C for 3 min.
5. Discard supernatant.
6. Resuspend the pellet in 200 μL of Reagent A (Mitochondrial
Isolation Kit for Tissue).
7. Vortex at maximum speed for 5 s.
8. Incubate samples on ice for 2 min.
9. Add 2.5 μL of Reagent B (Mitochondrial Isolation Kit for
Tissue).
10. Incubate on ice for 5 min and vortex for 5 s at every minute of
incubation.
11. Add 200 μL of Reagent C (Mitochondrial Isolation Kit for

Tissue).
12. Invert the tube for mixing, and then centrifuge at 3000 × g for
10 min.
13. Discard the pellet and keep the supernatant.
14. Centrifuge the supernatant at 3000 × g for 15 min.
15. Keep the resultant mitochondrial pellet and store supernatant
(see Note 3).
16. Wash the mitochondrial pellet with wash buffer (see Note 4).
17. Place samples on ice for lipid extraction.
18. Validate the mitochondrial isolation procedure by immunoblot
targeting for TOM20, a mitochondrial membrane protein, and
comparing with supernatant.
3.2 Butanol– 1. Resuspend mitochondrial pellets in 300 μL of n-butanol–

Methanol (BUME) Lipid methanol 3:1 (v/v).
Extraction 2. Vortex for 1 min.
3. Add 150 μL of heptane/ethyl acetate 3:1 (v/v) to the test tube.
4. Vortex for 1 min.
5. Add another 150 μL of heptane/ethyl acetate 3:1 (v/v) to the
microfuge tube.
6. Vortex again for 1 min.
7. Add 300 μL of 50 mM LiCl (see Note 3).
8. Vortex for 1 min.
9. Centrifuge the samples at 2700 × g for 10 min (see Note 4).
10. Collect the upper layer (organic layer) using a glass Pasteur
pipette, and place it into a newly labeled microfuge tube.
11. Repeat steps 1–9 two more times, collecting the organic layer
and combining it into the microfuge tube created in step 10.
12. Dry the organic layers using a SpeedVac Concentrator (see
Note 5).
13. Store the dried samples in a -80 °C until ready for mass
spectrometry analysis (see Note 6).
14. Perform a bicinchoninic acid (BCA) protein assay on the aque-
ous protein layer to normalize the lipid concentration in each
sample using the Pierce Micro BCA Protein Assay Kit.
3.3 High- 1. Resuspend dried lipid samples in 50 μL of chloroform/metha-

Performance Liquid nol 2:1 (v/v).
Chromatography and 2. Sonicate samples in an ultrasonic water bath for 20 min.
Mass Spectrometry
3. Vortex for 2 min. Briefly centrifuge until all the liquid has gone
(HPLC and MS) to the bottom of the microfuge tube.
Mass Spectrometry Analysis of Isolated Mitochondrial Lipids 5
4. Prepare and label LC-MS vials for both positive and

negative mode.
5. Take 25 μL of each sample and transfer it into the respective
LC-MS vial.
6. Analyze lipids by liquid chromatography electrospray tandem
mass spectrometry (LC-MS/ MS) using an Accucore™ Van-
quish™ C18+ Column (150 mm × 2.1 mm × 1.5 μm) and Q
Exactive Orbitrap Mass Spectrometer coupled to a heated elec-
trospray ionization source.
3.3.1 HPLC and MS 1. Solvents A and B consist of methanol/water 60:40 (v/v) with
Methods 10 mM ammonium acetate and methanol/chloroform 60:40
(v/v), respectively.
2. Run the gradient at 35–100% Solvent B for 13 min, hold at
35% solvent B for 2 min, and bring it back up to 100% solvent A
for 3 min and hold for 2 min (see Note 7).
3. Set the injection volume to 5 μL.
4. Set the HESI to a spray voltage of 4.415 kV, vaporization
temperature of 275 °C, and an auxiliary gas flow of 15 arbitrary
units.
5. Set the scan range at 150–1500 m/z.
3.4 Lipid 1. Upload raw data produced by LC-MS/MS to LipidSearch™.

Identification and 2. Set the parameters to the following:
Bioinformatics
• M-Score of 5.0.
Analysis
• Productsearch.
• Precursor (5/5) ppm.
• Intensity threshold of 1.0%.
3. Turn on quantitation and TopRank filter.
4. Grade ID quality from A-D.
5. Select all target classes.
6. Select all adducts for both negative and positive modes with the
exception of (CH3CH2)3NH+ and (CH3)2NH2.
7. Once all peaks are identified, align samples in positive mode,
negative mode, and also all trial runs in one.
8. Grade lipid identification from A–C with an M-Score of 5.
9. Export all aligned data for further analysis using the latest
version of MetaboAnalyst.
10. Normalize the data by pooling the control group, log trans-
forming, scaling by mean-centering, and dividing by the square
root of the standard deviation (pareto scaling).
4 Notes
1. This protocol was modified for use in small sample sizes. I

highly suggest performing a pilot study to determine appropri-
ate solvent amount for optimal extraction prior to performing
experiment with precious samples.
2. The addition of PBS is to remove all fat and debris surrounding
the optic nerve.
3. The supernatant can be stored for cytosolic analysis.
4. Follow your lab’s SOP for determining which wash buffer to
use. Depending on your lab’s SOP for a dot blot protocol,
TBS/TBS-T can be used.
5. Lithium chloride induces phase separation necessary to ade-
quately separate the lipids into the aqueous and organic layers.
6. After this step, the two layers should be visible so the upper
layer (organic layer) can be collected, and the bottom layer
(aqueous layer) can be re-extracted by phase separation
twice more.
7. Samples take anywhere from 30 to 90 min to completely dry.
For best practices, it is best to check on the samples after
30 min to assess dryness.
8. Flush samples with an inert gas (argon or nitrogen) if planning
for long-term storage (>1 week).
9. Prior to LC-MS/MS analysis, validate the method for mito-
chondrial lipid analysis using an internal standard with known
concentrations of cardiolipin.
References
1. Aufschnaiter A et al (2017) Mitochondrial of mitochondria from cell culture samples. Sci
lipids in neurodegeneration. Cell Tissue Res Rep 6:21107
367(1):125–140 7. Liao PC et al (2020) Isolation of mitochondria
2. D’Aquila P, Bellizzi D, Passarino G (2015) from cells and tissues. Methods Cell Biol 155:
Mitochondria in health, aging and diseases: 3–31
the epigenetic perspective. Biogerontology 8. Cruz M et al (2016) Improved butanol-
16(5):569–585 methanol (BUME) method by replacing acetic
3. Finsterer J (2011) Inherited mitochondrial acid for lipid extraction of biological samples.
neuropathies. J Neurol Sci 304(1–2):9–16 Lipids 51(7):887–896
4. Quintana-Cabrera R et al (2018) Who and 9. Lofgren L et al (2012) The BUME method: a
how in the regulation of mitochondrial cristae novel automated chloroform-free 96-well total
shape and function. Biochem Biophys Res lipid extraction method for blood plasma. J
Commun 500(1):94–101 Lipid Res 53(8):1690–1700
5. Ahmadpour ST et al (2020) Cardiolipin, the 10. Bligh EG, Dyer WJ (1959) A rapid method of
mitochondrial signature lipid: implication in total lipid extraction and purification. Can J
cancer. Int J Mol Sci 21(21):8031 Biochem Physiol 37(8):911–917
6. Kappler L et al (2016) Purity matters: a work-
flow for the valid high-resolution lipid profiling
Chapter 2
Isolation of Neuronal Synaptic Membranes by Sucrose

Gradient Centrifugation
Blake R. Hopiavuori, Dustin R. Masser, Joseph L. Wilkerson,
Richard S. Brush, Nawajes A. Mandal, Robert E. Anderson,
and Willard M. Freeman
Abstract
Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on
their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted
to specialized membranes. Without fractionation, these types of molecules could be below the levels of
detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution
throughout the various cell membranes. Isolation of specific membrane types where these lipids are
concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation
protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting
neural tissue homogenate: P1 (nuclear), P2 (cytoskeletal), P3 (neurosynaptosomal), PSD (post-synaptic
densities), and SV (synaptic vesicle).
Key words Sucrose gradient centrifugation, Membrane fractionation, Neuronal lipids, Synaptic
membrane isolation, Neurosynaptosome isolation
1 Introduction
Sucrose gradient centrifugation has been used by biochemists span-

ning the last several decades. It is an excellent approach for isolating
specialized lipid membranes based on their size and density. The
most common variations of this method are performed with either
a continuous or discontinuous gradient of various sucrose densities,
depending on the application. Discontinuous sucrose gradients
allowed the isolation of rod photoreceptor outer segments for
characterization of the rod photopigment, rhodopsin, and many
other specialized membrane proteins [1], as well as the large lipid
component of these membranes [2]. Redburn and Thomas first
described isolation of large (ribbon) and small (conventional)
synapses from rabbit retina [3]. Our group used a modified version

7
8 Blake R. Hopiavuori et al.
of Redburn’s protocol to resolve these large and small synapses

from outer segments in bovine retina to show, for the first time,
the presence of very long-chain (>26 C) polyunsaturated fatty acids
in the synapses of the neural retina [4, 5]. Herein we describe a
protocol for isolation of five major membrane fractions with clear
resolution from a starting homogenate of neural tissue that uses a
single density of 0.32 M sucrose and applies varying degrees of
centrifugal force on each supernatant to spin down each subsequent
fraction, a method which produces excellent resolution and enrich-
ment of synaptic membranes (Fig. 1). This is a modification to the
published work of VanGuilder et al. (2008 and 2010), in which this
type of sucrose gradient centrifugation was successfully used to
isolate neurosynaptosomes from diabetic rat retina and from aged
rat hippocampus, respectively, for transcriptomic and proteomic
analysis [6, 7]. As the fields of lipid biochemistry and neuroscience
continue to interact, these types of techniques will be paramount in
Fig. 1 Synaptic membrane isolation from fresh baboon hippocampus. (a) Representative electron micrographs
of all five fractions (P1, P2, P3, PSD, SV) plus the starting hippocampal homogenate (H). (b) Western blot
immuno-labeling for the synaptic vesicle-associated protein VGLUT2 shows dramatic enrichment in the
synaptic vesicle membrane fraction (SV) compared to the original starting homogenate and compared to
the P3 neurosynaptosomal fraction (15 μg of protein is loaded in each well; rabbit anti-VGLUT2 (Dcf68) 1:4000
was used followed by ECL anti-rabbit secondary 1:2000 for detection)
Isolation of Neuronal Synaptic Membranes 9
identifying new lipid molecules that, like docosahexaenoic acid and

arachidonic acid, are playing multifaceted roles in the central ner-
vous system.
2 Materials
Prepare all solutions fresh the day before use with ultrapure water
(u.p.H2O). Store at 4 °C (unless indicated otherwise). Follow all
waste disposal regulations when disposing of waste materials.
2.1 Solutions 1. 0.32 M sucrose/4 mM HEPES (300 mL): 32.86 g sucrose,

0.285 g HEPES, 3 mL 100 mM Na3VO4 (tyrosine phospha-
tase inhibitor; see below for making sodium orthovanadate),
pH to 7.4 with either HCl or NaOH as required; make to
300 mL with u.p.H2O.
2. Lysis buffer (15 mL): 0.09 g HEPES, 0.3 mL 0.05 M EDTA
(Ca2+ chelator), 0.088 g NaCl, 1.5 mL 10 mM DTT (sulfhy-
dryl reducing agent), 0.031 g NaF (phosphoseryl and phos-
phothreonyl phosphatase inhibitor), 150 μL Tween 20 (mild
detergent), 150 μL 100 mM Na3VO4, 2 Roche complete
EDTA-free protease inhibitor tablets, pH to 7.4 with either
HCl or NaOH as required; make to 15 mL with u.p.H2O.
3. Lysis water (20 mL): 20 mL u.p.H2O, 2 Roche complete
EDTA-free protease inhibitor tablets.
4. Post-synaptic density (PSD) resuspension buffer (15 mL):
75 μL 10% Triton-X 100 (mild detergent), 30 μL EDTA,
0.179 g HEPES, 2 Roche complete EDTA-free protease inhib-
itor tablets, pH to 7.4 with either HCl or NaOH as required;
make to 15 mL with u.p.H2O.
5. HEPES-NaOH (pH 7.4) to stop Lysis (1 M): 0.19 g HEPES
in 800 μL u.p.H2O, pH with NaOH to 7.4; make to 1 mL with
u.p.H2O.
6. 1 M Tris–HCl (pH 7.4) (1 L): 121.14 g Tris–HCl base in
800 mL u.p.H2O, pH to 7.4 with HCl; make to 1 L with u.
p.H2O.
7. Wash buffer (pH 7.4) (900 mL): 6 mL 1 M Tris–HCl (pH 7.4),
45 mL 2 M NaCl (prepared fresh), 1.8 mL 0.5 M EDTA, pH to
7.4 with HCl or NaOH; make to 900 mL with u.p.H2O.
8. Prepare a 100 mM solution of Na3VO4 (10 mL): adjust pH to
10.0 with NaOH, boil until solution is colorless, allow to cool
to room temp, repeat steps 1–3 until solution remains colorless
and pH is stable at 10.0, aliquot in 1 mL volumes and store at -
20 °C; thaw on ice on day of use.
2.2 Equipment 1. Centrifuge: Beckman Coulter Optima L-80 XP

Ultracentrifuge.
2. Rotors: Beckman SW 60 Ti swing-bucket rotor, SW 32 Ti
swing-bucket rotor.
3. Swing-buckets: SW 32 Ti (50 mL), SW 32.1 Ti (17 mL), SW
60 Ti (4 mL).
4. Centrifuge tubes: Beckman, Thinwall, Ultra-Clear centrifuge
tube 4 mL, 17 mL, or 50 mL (Catalog #344062, 344061,
344058).
3 Methods
As a general rule, 1 mL of buffer is used per 15–20 mg wet weight

of tissue, and this is considered one volume unit (see Notes 1 and
2). Always keep samples on ice, and pre-chill centrifuge and all
rotors to 4 °C before beginning. Euthanize rodents in accordance
with your institutional guidelines for animal care and use, keeping
in mind that CO2 and Halothane will rapidly change the structural
and functional integrity of neural membranes [8–11]. Our mice are
euthanized by cervical dislocation followed by decapitation; rats are
euthanized by decapitation. Our baboon brains were obtained
post-mortem on ice from the Department of Comparative Medi-
cine Primate Research Facility, University of Oklahoma Health
Sciences Center; hippocampus was dissected and processed using
this protocol. Due to the larger mass, the hippocampus from each
hemisphere was cut into three equal volumes and each of the six
pieces were processed in separate tubes before recombining like
samples at the very end before wash and resuspension (Fig. 1).
Perform all brain dissections on a cold aluminum plate on ice; use
a dissecting microscope to remove white matter, as the myelin will
disrupt the purity of the fractionation. Avoid attempting this frac-
tionation protocol on whole brain as there is a dramatic loss in the
quality of fraction resolution.
3.1 Isolation Steps 1. Place dissected tissue in one volume of ice-cold 0.32 M
sucrose/4 mM HEPES buffer in a Beckman, Thinwall, Ultra-
Clear centrifuge tube 4 mL, 17 mL, or 50 mL, depending on
tissue weight (1 volume = approx. 1 mL buffer per 15–20 mg
tissue wet weight) (see Notes 1 and 2).
2. Incubate on ice for 20 min (allow tissue to sink to the bottom).
Pour off buffer, and replace it with one volume of ice-cold
0.32 M sucrose/4 mM HEPES buffer, and incubate on ice
for another 20 min.
3. Invert tube 2–3 times and incubate on ice for another 10 min.
4. After tissue settled to the bottom of the tube, replace buffer

with one volume of fresh ice-cold 0.32 M sucrose/4 mM
HEPES buffer and repeat step 3.
5. Replace the buffer with one volume of fresh ice-cold 0.32 M
sucrose/4 mM HEPES buffer and pour into homogenizer. Be
sure to leave enough room in the tube to accommodate the
homogenization pestle.
(a) With a motorized Dounce pestle, keeping the samples on
ice (speed setting = 1+), use slow, up and down motion,
to homogenize tissue using the full range of motion avail-
able and holding for ~3 s at the bottom of the tube (5–10
times).
(b) With a glass Teflon hand homogenizer, keeping the sam-
ples on ice, tissue is homogenized with 10–15 slow, even,
up and down strokes until the tissue has become homog-
enous; apply pressure and twist pestle back and forth at
the bottom of each stroke to better break up the tissue.
* See Note 3 before continuing.
6. Transfer back to the Beckman ultracentrifuge tube, and place
them in the pre-chilled rotor, making sure that all tubes are
balanced by weight (we use the following combination of Beck-
man swinging bucket rotors: SW 28.1, SW 32.0 Ti, SW 32.1
Ti, SW 60 Ti).
7. Spin 1 (RCF = 200 × g) (see Note 4 re: RCF) at 4 °C for
10 min to isolate the P1 nuclear fraction (this is a very
low-speed spin so be careful not to resuspend the pellet while
removing the supernatant).
8. Place P1 pellet on ice and transfer supernatant to a new Beck-
man ultracentrifuge tube and balance by weight for the
next spin.
9. Spin 2 (RCF = 800 × g) at 4 °C for 12 min to isolate the P2
cytoskeletal fraction.
10. Place P2 pellet on ice and transfer supernatant to a new Beck-
man ultracentrifuge tube and balance by weight for the
next spin.
11. Spin 3 (RCF = 25,000 × g) at 4 °C for 14 min to isolate the
P3 neurosynaptosomal fraction.
12. Spin 4: Discard the supernatant from your P3 pellet (myelin,
microsomes, etc.), or keep for analysis if desired. Wash the P3
pellet in a fresh volume of ice-cold 0.32 M sucrose/4 mM
HEPES buffer, balance by weight, and spin again at
RCF = 25,000 × g at 4 °C for 12 min; discard this
supernatant.
STOP: At this stage you have isolated your neurosynapto-

somes and can stop here if you do not need to perform the further
fractionation required to isolate PSDs and SVs; you may keep the
final supernatant from your P3 spin (myelin, microsomes, etc.) for
analysis or discard and place your P3 pellet on ice. See Subheading
3.2 for final wash protocol for all pellets [OR] if you require
enrichment of PSD and/or SVs; then continue to step #13.
* See Note 3 before continuing. Your P3 pellet is now your
new starting material.
13. Lyse P3 pellet by resuspending in 3.6 mL ice-cold lysis water
+0.4 mL ice-cold 0.32 M sucrose/4 mM HEPES buffer (see
Note 5) and after transferring to homogenization tube,
homogenize by either method (a) or method (b) using only
3–5 strokes this time.
14. Rapidly add 37.5 μL of the ice-cold HEPES–NaOH (pH 7.4)
buffer and incubate for 30 min on ice to stop lysis.
15. Spin 5: balance tubes by weight and spin again to pellet at
RCF = 25,000 × g at 4 °C for 20 min.
16. Transfer the supernatant from spin 5 to a new tube and set on
ice for later (SVs fraction isolation).
17. Resuspend the pellet from spin 5 in 3 mL PSD resuspension
buffer and balance tubes by weight for the next spin.
18. Spin 6 (RCF = 32,000 × g) at 4 °C for 20 min to isolate the
PSD post-synaptic density fraction. Place this pellet on ice
until ready to begin final wash protocol. Discard supernatant
(myelin, microsomes, etc.) or keep for analysis if desired.
19. Take the supernatant from spin 5 that you set aside earlier and
balance tubes by weight for the next spin with a pre-combined
3.6 mL lysis water +0.4 mL 0.32 M sucrose/4 mM HEPES
solution.
20. Spin 7 (RCF = 165,000 × g) at 4 °C for 120 min. to isolate
the SV synaptic vesicle fraction in the pellet. Discard super-
natant or store for analysis if desired.
3.2 Wash and 1. Resuspend all pellets (P1 through SV) that have been kept on
Storage Steps ice until now in one volume each of ice-cold wash buffer. Do
the same for your starting homogenates as well (H alone or H
and P3 if you continued all the way through to SV isolation);
dilute with wash buffer to remove sucrose.
2. Balance tubes by weight. If there is more than one tube for
each fraction for the same biological replicate, now is the time
to combine them before moving on (see Note 6).
3. Spin 8 (RCF = 25,000 × g) at 4 °C for 20 min to remove all
excess sucrose. For washing SV use RCF = 160,000 × g.
Discard supernatants.
4. Resuspend final pellets in an appropriate volume of wash buffer

+ one Roache protease inhibitor table (P.I.) per 10 mL of wash
buffer (see Note 7). Base the volume used on the size of the
pellet; SVs will usually require the smallest volume and P1/P2
the largest (typically the range falls between 100 and 900 μL).
This will depend on the amount of starting material, yield
obtained, and end-point experiments. We set aside a portion
of this final suspension for membrane lysis and protein isolation
for western blotting and use the rest for various lipidomic
analyses.
5. Transfer into cryo-vials, and snap-freeze in liquid nitrogen, and
store at -80 °C until ready for lipidomic and proteomic
analyses.
4 Notes
1. The volume unit can be adjusted if needed; lowering the tissue

weight closer to 10–15 mg/mL may improve separation.
Remember that in some cases, the tissue may be too large to
combine all of the tissue from the same biological replicate into
one ultracentrifuge tube. If this happens, the tissue can be
blocked into more appropriate sizes and processed in more
than one tube within the same centrifuge. In this case, at the
very end, be sure to combine like fractions from the same
biological replicate before doing your final wash spin to save
yourself time and to improve your final yield.
2. Always keep an accurate account of your working volumes. The
scale of the experiment can vary dramatically when comparing
the volumes needed to isolate synaptosomes from baboon
hippocampus vs. that of a mouse so plan accordingly and
think through each step so that you do not get caught with
either too large or too small a volume.
3. Always remember to set aside your starting material. It is not
sufficient to use dissected but un-fractioned tissue as a repre-
sentation of what you started with. You should set aside an
aliquot of your starting homogenate (H) before you proceed to
spin 1 and again from your P3 pellet as a new starting material
before lysing for PSD and SV isolation. These aliquots will
remain homogenous on ice during the experiment and will be
spun down later during the wash phase into a pellet for resus-
pension and snap-freezing.
4. Note that RCF is equal to the gravitational force, and for each
rotor that you plan to use, you will need to calculate the RPMs
that correspond to the RCF values reported here. Be sure to
balance tubes (by weight) that will oppose each other in your
rotor. We perform all fractionation with swinging bucket rotors
as they seem to produce a better yield with better resolution
than the fixed-angle rotors.
5. Note that in step 13, 4 mL is being added in total to each P3
starting pellet (3.6 mL of lysis water +0.4 mL of 0.32 M
sucrose/4 mM HEPES). We use these volumes because our
centrifuge tubes for the SW 60 Ti only hold a volume of 4 mL;
if you are able to achieve 160,000 × g with tubes of a larger
volume, simply increase the total volume while keeping the
ratio between the two buffers the same. Be sure that protease
inhibitors are present in your lysis water. Be careful not to over-
homogenize here; do not use more than five complete strokes
with either method of homogenization.
6. Washing will require several spins to work through all of the
samples depending on how many systems you are able to use at
once, your number of replicates, and how many fractions you
have generated.
7. When resuspending your final pellets for snap-freezing,
remember that it is always easier to dilute your sample further
than it is to concentrate it down, so, if in doubt, stick to the
smaller side when choosing your resuspension volumes.
Acknowledgments
Heather VanGuilder Starkey for sharing her isolation protocols

with us. Nicolas Bazan for sending us the rabbit anti-VGLUT2
antibody (Dcf68) cloned by his laboratory.
This work was supported by the following grants: EY024520,
EY021716 to WMF; EY023202 to DRM; NS090117,
EY004149, EY000871, and EY021725 (P30 Vision Core Grant)
to REA; NS089358 to BRH; EY022071, EY025256, to NAM; and
Foundation Fighting Blindness to REA and NAM. Unrestricted
grant from Research to Prevent Blindness, Inc.
References
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rhodopsin. J Mol Biol 282(5):991–1003. deficient photoreceptor terminals. Invest
https://doi.org/10.1006/jmbi.1998.2070 Ophthalmol Vis Sci 55(7):4063–4072.
2. Anderson RE, Maude MB (1970) Phospholi- https://doi.org/10.1167/iovs.14-13997
pids of bovine outer segments. Biochemistry 5. Hopiavuori BR, Bennett LD, Brush RS, Van
9(18):3624–3628 Hook MJ, Thoreson WB, Anderson RE (2016)
3. Redburn DA, Thomas TN (1979) Isolation of Very long-chain fatty acids support synaptic
synaptosomal fractions from rabbit retina. J structure and function in the mammalian ret-
Neurosci Methods 1(3):235–242 ina. OCL 23(1):D113
6. VanGuilder HD, Brucklacher RM, Patel K, 9. Martoft L, Stodkilde-Jorgensen H, Forslid A,

Ellis RW, Freeman WM, Barber AJ (2008) Pedersen HD, Jorgensen PF (2003) CO2
Diabetes downregulates presynaptic proteins induced acute respiratory acidosis and brain
and reduces basal synapsin I phosphorylation tissue intracellular pH: a 31P NMR study in
in rat retina. Eur J Neurosci 28(1):1–11. swine. Lab Anim 37(3):241–248. https://doi.
https://doi.org/10.1111/j.1460-9568.2008. org/10.1258/002367703766453092
06322.x 10. Mikulec AA, Pittson S, Amagasu SM, Monroe
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WE, Freeman WM (2010) Aging alters the action potential conduction in hippocampal
expression of neurotransmission-regulating axons. Brain Res 796(1–2):231–238. https://
proteins in the hippocampal synaptoproteome. doi.org/10.1016/S0006-8993(98)00348-5
J Neurochem 113(6):1577–1588. https://doi. 11. Silva JH, Gomez MV, Silva JH, Guatimosim C,
org/10.1111/j.1471-4159.2010.06719.x Gomez RS (2008) Halothane induces vesicular
8. Angus DW, Baker JA, Mason R, Martin IJ and carrier-mediated release of [3H]serotonin
(2008) The potential influence of CO2, as an from rat brain cortical slices. Neurochem Int
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of basic compounds in rodents. Drug Metab j.neuint.2008.01.004
Dispos 36(2):375–379. https://doi.org/10.
1124/dmd.107.018879
Chapter 3
Multi-macromolecular Extraction from Endosymbiotic

Anthozoans
Abstract
Obligately symbiotic associations between reef-building corals (anthozoan cnidarians) and photosyntheti-
cally active dinoflagellates of the family Symbiodiniaceae comprise the functional basis of all coral reef
ecosystems. Given the existential threats of global climate change toward these thermo-sensitive entities,
there is an urgent need to better understand the physiological implications of changes in the abiotic milieu
of scleractinian corals and their mutualistic algal endosymbionts. Although initially slow to leverage the
immense breakthroughs in molecular biotechnology that have benefited humankind, coral biologists are
making up for lost time in exploiting an array of ever-advancing molecular tools for answering key questions
pertaining to the survival of corals in an ever-changing world. In order to comprehensively characterize the
multi-omic landscape of the coral holobiont—the cnidarian host, its intracellular dinoflagellates, and a
plethora of other microbial constituents—I introduce a series of protocols herein that yield large quantities
of high-quality RNA, DNA, protein, lipids, and polar metabolites from a diverse array of reef corals and
endosymbiotic sea anemones. Although numerous published articles in the invertebrate zoology field
feature protocols that lead to sufficiently high yield of intact host coral macromolecules, through using
the approach outlined herein one may simultaneously acquire a rich, multi-compartmental biochemical
pool that truly reflects the complex and dynamic nature of these animal–plant chimeras.
Key words Coral reefs, Gene expression, Mass spectrometry, Multi-omics, Proteomics, Symbiosis
1 Introduction
1.1 Safety High-efficiency extraction of RNAs, DNAs, proteins, lipids, and

Considerations polar metabolites from endosymbiotic anthozoans warrants the use
of toxic, corrosive chemicals, namely, an acid–guanidinium+phe-
nol-based solvent (e.g., TRIzol®, Thermo-Fisher Scientific [TFS])
and chloroform. All work should be undertaken while wearing
nitrile gloves, a laboratory coat, safety goggles, and closed-toe
shoes. All steps should be performed in a fume hood except for
those associated with the RNA/DNA spin column protocols,
which can be performed on a standard laboratory benchtop. Dis-
pose of all organic solvent waste as recommended by your local

17
18 Anderson B. Mayfield
Occupational Health and Safety Administration (OSHA). I recom-

mend that even plastics and glassware that have come into contact
with the aforementioned chemicals be treated as hazardous
material.
1.2 Drawbacks to The extraction and subsequent purification of the major cellular
Current Approaches macromolecular species—DNAs, RNAs, proteins, lipids, and other
(polar) metabolites—from reef-building corals (Fig. 1), endosym-
biotic sea anemones, and their dinoflagellate endosymbionts
(Fig. 2) is still in its infancy, despite publications on these topics
dating back into the 1980s (e.g., [1, 2]). This statement would
appear to be at odds with the fact that many hundreds of peer-
reviewed articles have been produced on coral gene expression
alone (e.g., [3–6]). Has our field reached the state-of-the-art level
of analysis that was once only realized by those working on model
organisms, such as mice? Unfortunately, this is not yet the case. For
one, over 10 years ago, Mayfield et al. [7] revealed that the vast
majority of reef coral and endosymbiotic sea anemone (which
can serve as a model system for the more-difficult-to-study corals
[8, 9]) articles had either completely failed to extract macromole-
cules from the dinoflagellate endosymbionts (family Symbiodinia-
ceae) or, if they were extracted, they were left unanalyzed (or treated
as contamination). The failure to either extract dinoflagellate
biological material or include Symbiodiniaceae data in molecularly
focused publications of that era (e.g., [10]) potentially left many to
conclude that these algae, which are obligate, photosynthetic
mutualists without which reef-building corals would perish from
Fig. 1 An Indo-Pacific coral reef featuring a high abundance of a branching coral

species (Acropora sp.). (Photo by the author)
Marine Multi-Omics 19
Fig. 2 A scanning electron micrograph of decalcified, freeze-fractured reef coral

(Pocillopora sp.) tissues. The spherical objects (~10 μm in diameter) in the
gastrodermal (right-most) tissue layer represent the dinoflagellate
endosymbionts (family Symbiodiniaceae)
starvation [11], are “background players” with respect to their

contribution to the macromolecular pool emerging from coral
tissue biopsies; this uni-compartmental focus remains regrettably
prevalent to this day (Table 1), and very few studies have made an
effort to profile macromolecular species from multiple members of
the coral “holobiont” (the collective name for the association of
coral, dinoflagellates, and other eukaryotic and bacterial microbes
that associate with the anthozoan host). My primary goal in this
chapter is to challenge marine biologists to attempt to more com-
prehensively characterize the complex molecular landscapes of their
target species; it may result in a more exhausting day in the labora-
tory, but I hope to convince you that it is worth the extra effort.
Although it is certain that the intracellular dinoflagellate popu-
lations do not contribute more biomass to the holobiont than the
host anthozoans in which they reside [12, 13], when using proper,
robust extraction techniques, they can actually yield as much, if not
more, of the target macromolecules of interest. Perhaps this is
unsurprising. After all, dinoflagellates have amongst the largest
genomes ever characterized [14], meaning that, even if there
were, for instance, five times more host tissue material than dino-
flagellate, if the latters’ genomes are truly five times larger than
those of the average coral (a good current estimate), a similar
amount of host coral and Symbiodiniaceae DNA might very well
be extracted from the same biopsy; in fact, this does indeed appear
to be the case [15].
Table 1
20
Simple-random-sample of 20 endosymbiotic anthozoan molecular biology articles
Molecular
Molecules Considered Considered dino- Host/symbiont integrity
Reference characterized Extraction approach anthozoan host? flagellates? molecular ratio reported?
Mayfield [40] RNA, DNA, TRIzol® + mor tar and pestle Yes Yes ~2:1 Yes
protein
Mayfield & RNA, DNA TRIzol® + mor tar and pestle Yes Yes ~2:1 Yes
Dempsey
[45]
Rubin et al. [46] RNA NucleoSpin® TriPrep+mor tar Yes Yes ~1:2 No
and pestle+bead mill
McRae Protein TRIzol® + mor tar and pestle Yes Yes ~14:1 Yes
et al. [47]a
Tisthammer Protein Urea+tissue shearer Yes No NA No
et al. [48]
Sproles et al. Protein, DNA LN2 + mechanical Yes No NR No
[49]b homogenization
Rocker RNA, lipid Aurum™ Total RNA minikit, Yes No NA No
et al. [50] 2:1 dichloromethane:methanol
Dimos RNA RNAqueous® Yes No NA Yes
et al. [51]
Lohr et al. [52] Metabolites Methanol Yes No NA No
Wright RNA RNAqueous® + bead mill Yes Yes ~9:1 No
et al. [53]
Seveso Protein SDS buffer+mor tar and pestlec Yes No NA No
et al. [54]
Chen et al. [55] Lipid, protein Bligh and Dyer [56] Yes Yes NR No
Ricaur te et al. Protein Rehydration buf fer+mor tar and Yes No NA Nod
[57] pestle
Vidal-Dupiol RNA TRIzol® (details not repor ted) Yes No NA No
et al. [58]
Barshis et al. RNA TRIzol® + bead mill Yes No NA No
[59]
Putnam et al. RNA, DNA, TRIzol® + mor tar and pestle Yes Yes ~2:1 Yes
[60] protein
Mayfield et al. RNA, DNA, TRIzol® + mor tar and pestle Yes Yes ~2:1 Yes
[16] protein
Kenkel RNA RNAqueous® + razor blade Yes No NA Yes
et al. [61]
Mayfield et al. RNA, DNA TRIzol® + mor tar and pestle Yes Yes ~2:1 Yes
[15]
Mayfield et al. RNA, DNA TRIzol® + mini-pestle Yes Yes Variableb,e Yes
[7]
Unless other wise noted, all studies were undertaken with adult scleractinian corals. All trademarked reagents and kits are from Thermo-Fisher Scientific except NucleoSpin®
(Macher y-Nagel) and Aurum™ Total RNA minikit (Bio-Rad)
NA not applicable, NR not repor ted
a
Analyzed both adult and lar val corals
b
Used endosymbiotic sea anemones
c
Erroneously claims to be a dinoflagellate-free preparation method (not demonstrated)
d
Proteins appear degraded on the gel images presented
e
Depended on infection status of anemones
Marine Multi-Omics
21
While genome size is static within a living cell, it is more

difficult to quantify or model a “typical” host coral or Symbiodi-
niaceae transcriptome, proteome, lipidome, or metabolome
because the respective molecules are in a constant state of flux due
to the metabolic needs of the cells. In the few instances in which the
relative host coral/Symbiodiniaceae mRNA ratios were reported
(e.g., [16]; Table 1), it is not uncommon for the dinoflagellates to
contribute over 1/3 of all extracted RNA in a healthy reef coral.
Those looking to model the health of a coral, then, would be wise
to include the dinoflagellate contigs in their bioinformatic analyses
[17], though to this date, the more common reductionistic
approach is taken; researchers interested in host coral gene expres-
sion omit the Symbiodiniaceae genes, while others exclusively
focused on the endosymbionts omit those genes of their hosts
(Table 1). Although this approach is sensible for certain laboratory
experiments, I argue that, for coral biologists seeking to predict the
ultimate fates of reef-building corals (e.g., [18–21]), such a sim-
plistic approach is decidedly sophomoric. Instead, those interested
in characterizing the health and stress tolerance of corals using
molecular approaches (sensu [22]) would do far better by consider-
ing all constituents of the coral holobiont or at least the coral host
and endosymbiotic dinoflagellates at a minimum.
The overall tendency to exclude Symbiodiniaceae molecules
from omics analyses, however, may not actually stem from a scien-
tific desire to focus exclusively on one member of the association. I
surmise herein that it actually follows just as much from poor-
quality extraction techniques, or, potentially more likely, the use
of extraction protocols originally optimized for use with model
organisms such as E. coli. Were the latter to be the case, one
might actually expect a host coral-dominated macromolecular
extract. To understand why this is the case, a more detailed under-
standing of coral-dinoflagellate biology is warranted. Firstly, reef-
building corals and endosymbiotic sea anemones (e.g., Exaiptasia)
house the Symbiodiniaceae dinoflagellates in only half of their cells,
those of the gastrodermal tissue layer [23]. In essence then, the
coral/endosymbiotic anemone is an animal–plant chimera. The
anthozoan host cells feature a cell membrane that entirely ensheaths
the endosymbionts (Fig. 2); the endosymbionts are not swimming
freely within an open space, as are the bacterial microbes in our
guts, but are effectively trapped in what is known as a “symbio-
some” [24]. From an extraction standpoint, this does not pose an
issue because the anthozoan cell membranes are decidedly flimsy
and are easily disrupted by manual or chemical agitation [25]. In
fact, were one to immerse a coral polyp in freshwater, many antho-
zoan cells would lyse due to osmotic stress alone [26], without any
need for corrosive solvents, bead mills, mortars and pestles, etc.
Where technique becomes more critical, though, is in the lysing

of the dinoflagellate cells, which are surrounded by some of the
strongest cell walls ever before studied [27]. In the hypothetical
example above of freshwater lysis, it is doubtful that any Symbio-
diniaceae cells would burst. This is not to say that they are resistant
to osmotic stress, only that their hardy cell walls would prevent
lysis. Extraction buffers featuring even guanidinium-based salts
(found in virtually all commercially available spin column kits)
and beta-mercaptoethanol may even be insufficient to thoroughly
lyse the majority of Symbiodiniaceae cells in a coral tissue biopsy
and are only useful when employed with vigorous mechanical agi-
tation (e.g., a bead mill with acid-washed sand; [28]). For those
researchers aiming to characterize only host anthozoan biomolecu-
lar material, this is actually advantageous; one could realistically
expect to extract only host macromolecules using these “gentler”
extraction approaches. Within seconds of immersion in
guanidinium-based lysis buffers (or sodium dodecyl sulfate [SDS]
at 2% or greater), the vast majority of anthozoan cells will lyse,
freeing the cellular macromolecules into solution to be concen-
trated, purified, and characterized as outlined by the protocols
described herein.
However, those like myself who seek to also analyze the macro-
molecules encumbered within the dinoflagellate cells must unfor-
tunately use a more time-consuming, rigorous extraction approach
to ensure that the incredibly resistant algal cell walls are destroyed.
In other words, the extraction approach outlined in this chapter is
best suited for researchers seeking to characterize RNA, DNA,
proteins, lipids, and metabolites from host corals (or anemones),
endosymbiotic dinoflagellates, and the myriad other microbes that
call the polyp, coenosarc, or colony home (Fig. 3). A far simpler and
Fig. 3 A schematic featuring a brain coral (~50 cm in diameter) and a depiction

of the multi-omic approach presented herein. Note that, in the Venn diagram on
the right, “metabolomics” includes both polar metabolites and lipids
safer approach featuring commercially available spin columns

would be better suited for biologists with an interest in host antho-
zoan biological material only (not described herein).
1.3 Justification for Although several exceptions abound (Table 1), another general
a Multi-omic Approach drawback of many endosymbiotic anthozoan studies, and likely
nearly all biological disciplines for that matter, is the extraction of
only a singular macromolecular type from each biopsy. One might
ask, “I have plentiful tissue with which to work; can I not simply
extract RNA from biopsy A, DNA from biopsy B, lipids from
biopsy C, etc?” For certain experiments and tissues types, this
might very well be the most practical solution. Certainly, if one is
interested in gene expression alone, for instance, why bother to
extract the other cellular macromolecules? However, for reef-build-
ing corals, a multi-omic approach is recommended for at least two
reasons. First, numerous reef-building corals associated with multi-
ple dinoflagellate endosymbiont types [29], not to mention a
plethora of other microbes [30]. Even if one were only interested
in functionally profiling lipids, metabolites, mRNAs, or proteins,
there is still good reason to co-extract, at minimum, the DNAs such
that these complex microbial communities could be analyzed in
tandem. Indeed, this is exactly what was done in the first multi-
omic analysis of a reef coral [16].
Second, although gene expression research is far more popular
these days given growing dataset sizes and diminishing sequencing
costs, there is no correlation between anthozoan or Symbiodinia-
ceae mRNA levels and concentrations of the proteins they encode
in the few studies in which such congruency has been experimen-
tally tested [31–34]. As such, those looking to understand the
physiological implications of climate change or other environmen-
tal changes on reef corals and their diverse microbial communities
should at least (1) profile protein concentrations and (2) character-
ize the identity of the holobiont from genetic or even meta-
genomic co-analysis of the DNAs. More generally, then, reef coral
biologists are among the most justified in employing a multi-omic
approach in their research projects.
1.4 Protocol Analysis of complex suites of macromolecules—namely, RNAs,

Overview and DNAs, proteins, lipids, and polar metabolites—of biological speci-
Rationale mens involves the following steps (Fig. 4): (1) preservation of the
biological specimen (i.e., biopsy), (2) extraction of target macro-
molecules from the cells, (3) separation of the macromolecules
from one another, (4) concentration of the macromolecules,
(5) washing of the macromolecules, and (6) solubilization of the
macromolecules. Although myriad options abound, I recommend
and will consequently describe two over-arching approaches:
Fig. 4 An overview of the multi-omic extraction and representative coral sample material. For scleractinian
corals with large corallites (e.g., those of the Montastraea cavernosa colony in this figure), a single polyp will
yield sufficient RNA, DNA, protein, lipid, and metabolite quantities for all manner of omic analyses. Small-
polyped corals (~1 mm, e.g., pocilloporids) may instead necessitate 15–30 polyps to yield sufficient protein, in
particular
Option A (Fig. 5) and Option B (Fig. 6). The former is superior in

that the proteins are far higher in quantity relative to Option B. The
major disadvantage of Option A is that polar metabolites are
co-isolated with RNAs and DNAs and must be subsequently
pooled, then separated from one another, concentrated, and pur-
ified. This results in a large number of steps, as well as many dozens
of microcentrifuge tubes to track. In contrast, lipids and polar
metabolites are easily partitioned with a modified Bligh and Dyer
extraction in Option B, which is derived from a seminal work by
Podechard et al. [35]. However, in this approach, the proteins form
a lens between the lipids and polar metabolites, and it is difficult to
re-dissolve these irreversibly denatured species. They may, then,
only be suitable for normalizing lipid and metabolite levels to
total protein and not for explicit proteomic analyses; in fact, I
have never used the proteins from Option B for shotgun or label-
based proteomics (both described below).
With the exception of the proteins of Option B, these protocols
consistently yield high-quantity and high-quality macromolecules
from a diverse array of marine organisms, though they have only
ever been thoroughly optimized for reef-building corals (both
larvae lacking in skeletal material and adult corals featuring large
quantities of calcium carbonate skeleton) and model anthozoan–
dinoflagellate endosymbioses, namely, Exaiptasia spp. Differing
sample types may require slight adjustments.
Fig. 5 Endosymbiotic anthozoan multi-omic extraction approach-Option

A. Please note that the cartoon coral polyp has been magnified approximately
ten-fold relative to the adjacent cartoon of the microcentrifuge tube. Also note
that it is possible that a new solvent system could be exploited to better separate
lipids from polar metabolites (between steps 4 and 5a)
2 Materials
Those familiar with more traditional phenol–chloroform DNA

extractions will recognize the vast majority of reagents and materi-
als required for successful extraction of macromolecules from reef-
building corals and endosymbiotic sea anemones. Please take note
of the safety concerns outlined previously, particularly with the
handling of TRIzol (TFS) and chloroform. With four exceptions
(denoted by asterisks), all chemicals found in the list below can be
purchased directly from your preferred vendor. For solvents, I have
noted the preferred quality grade in parentheses (when applicable),
and for less common chemicals, I have mentioned a representative
manufacturer; never use ACS or “reagent-grade” chemicals. When
a chemical/reagent name has been abbreviated from hence forth,
the abbreviation is shown as the first word within the parentheses.
Option B Podechard et al. (35)

Bligh & Dyer
extraction
RNA A single sample
DNA
Protein RNA extraction Polar
Lipids metabolites
Polar matabolites Organic phase
Aqueous phase : RNA
• Same as Option A except: Organic phase : Lipids

Lipids
Bligh & Dyer

• Instead of adding acetone to proteins,
carry out a Bligh and Dyer* extraction Lipid Analysis
using the organic phase containing polar

proteins & lipids. protein layer
non-polar
• Pros: better separates lipids from other A. cervicornis

Andersson et al. (43)
metabolites.
polar
protein layer
• Cons: the protein is far lower quality non-polar
and quantity. coral skeleton
*Methanol+chloroform+water
Fig. 6 Endosymbiotic anthozoan multi-omic extraction approach-Option B. Please note that these images have
been modified from the respective publications (both of which are open access and cited in the reference list
[35, 43]). A. cervicornis = Acropora cervicornis (a reef-building coral that is the primary focus of reef
restoration efforts in South Florida, USA)
1. Acetone (molecular-grade; 100%; numerous vendors).

2. Acetonitrile (ACN; 100%; HPLC-grade; numerous vendors).
3. Agarose (powder; molecular-grade; numerous vendors).
4. Ammonium bicarbonate (AB; e.g., Sigma-Aldrich). Only buy
in powdered form as solutions are unstable.
5. Autoclave.
6. Back extraction buffer* (BEB): 1 M Tris base, 4 M guanidi-
nium (iso)thiocyanate, and 50 mM sodium citrate (Na citrate):
(a) This near-saturated buffer is, in my experience, the only
means of co-extracting high molecular weight DNA from
samples stored in TRIzol; using TFS’ DNA extraction
protocol instead leads to recovery of DNA of far lower
molecular weight.
(b) Guanidinium thiocyanate is unstable in solution, even
when kept in the dark; make only enough for several
weeks’ worth of sample processing (you will need
0.5 mL/sample.).
(c) Place a stir bar in the glass bottle you will use and add a
small amount of deionized+distilled water (ddH2O).
(d) Weigh out the appropriate quantity of guanidinium thio-
cyanate and add to bottle.
(e) Add a small amount of water to coat the guanidinium

thiocyanate in the bottle.
(f) Weigh out additional solutes (Tris base and Na citrate)
and add to bottle.
(g) Normally, you will not need to add much excess water; the
guanidinium thiocyanate accounts for the majority of the
BEB volume.
(h) For this reason, do not add a large amount of water to the
bottle first, or you will far surpass your target volume
upon addition of the guanidinium thiocyanate.
(i) Given the large amount of solutes, several hours on a stir
plate may be required to fully solubilize all components;
make the BEB the day prior to starting your extractions.
(j) Wrap bottle in aluminum foil and store in the dark.
(k) Periodically check coloration; once solution has turned
yellow, discard (as hazardous material) and make a new
batch.
7. Balance with a precision of at least 1 mg.
8. Beads (steel or ceramic) for bead mill (optional; numerous
vendors).
9. Bead mill (optional; e.g., MP Biomedical’s FastPrep™ series).
10. Beta-mercaptoethanol (100% solution; hazardous; numerous
vendors).
11. Bovine serum albumin (BSA; numerous vendors). Can buy in
lyophilized form or pre-solubilized (in which it is important
that a preservative has been added).
12. Butylated hydroxytoluene (BHT; numerous vendors).
13. C18 spin tips (optional; Pierce).
14. Centrifuge capable of speeds up to 12,000 × g for 1–2 mL
microcentrifuge tubes and 1500 × g for 15 mL tubes.
15. Chloroform (molecular-grade; amylene-free; hazardous;
numerous vendors).
16. Culture tubes–glass with Teflon-coated plastic lids (15 mL;
e.g., Pyrex®).
17. Culture tube racks.
18. Diethyl pyrocarbonate (DEPC; numerous vendors):
(a) DEPC is highly carcinogenic.
(b) Those with the means to do so should consider purchas-
ing nuclear-free water, rather than performing in-house
DEPC treatments (not discussed herein).
19. Dithiothreitol (DTT; note that this is a poison with a noxious
smell; numerous vendors).
20. DNA gel stain: SYBR® Red or SYBR® Gold (TFS)—avoid

using ethidium bromide.
21. DNA molecular weight ladder (numerous vendors, e.g.,
Bio-Rad).
22. DNA spin column kit (optional, e.g., Qiagen).
23. Ethanol (100%; molecular-grade [nuclease-free]; numerous
vendors).
24. Ethylenediaminetetraacetic acid (EDTA; e.g., Sigma-Aldrich).
25. Formic acid (numerous vendors).
26. Fume hood.
27. Gel doc+power supply for agarose gel electrophoresis.
28. Gel doc+power supply for SDS-PAGE (e.g., Phastgel®).
29. Glycerol (100% stock; nuclease-free; numerous vendors).
30. Graphite spin columns (optional; Pierce).
31. Guanidine hydrochloride (HCl; hazardous; numerous
vendors).
32. Guanidinium thiocyanate (aka isothiocyanate; hazardous;
numerous vendors).
33. High salt solution* (HSS): 0.8 M Na citrate and 1.2 M sodium
chloride (NaCl) in DEPC-treated water:
(a) Unlike the BEB, the HSS is non-toxic, easy-to-make, and
the solutes dissolve fully within several minutes.
(b) HSS is stable for many weeks at room temperature,
though precipitation of salts in this near-saturated solu-
tion is inevitable:
(i) Consider making small quantities (5–10 mL; enough
for 20–40 samples at 0.25 mL/sample).
(ii) Limit opening of the bottle, which can allow RNases
to enter.
(c) Weigh out appropriate quantities of NaCl and Na citrate
and add to DEPC-treated or otherwise nuclease-free
water.
(d) Vortex vigorously and allow several minutes for salts to go
into solution.
34. Hydrochloric acid (HCl; hazardous; numerous vendors).
35. Inert gas (argon or nitrogen).
36. Iodoacetamide (powder; numerous vendors).
37. Isobaric tags for relative and absolute (protein) quantification
(iTRAQ; Sciex).
38. Isopropanol (100%; molecular-grade [nuclease-free]; numer-
ous vendors).
39. Kim® Wipes (for drying samples in inverted microcentrifuge

tubes on benchtop).
40. Laboratory coat.
41. Laemmli sample buffer: 2% sodium dodecyl sulfate, 2% beta-
mercaptoethanol, 10% glycerol, and 0.0625 M Tris-HCl
(pH 6.8).
42. Liquid chromatography (LC) column (e.g., Acclaim™ Pep-
Map™ RSLC [75 μm × 15 cm] nanoViper column [TFS]).
43. Lipid standards (optional; e.g., Avanti SPLASH®
LIPIDOMIX®).
44. Liquid nitrogen (LN2; optional).
45. Liquid nitrogen vapor (i.e., “dry”) shipper (optional).
46. Mass spectrometer (MS; e.g., Orbitrap/Q Exactive [TFS]).
47. Metabolite standards (optional; e.g., IROA’s suite of
pre-mixed standards).
48. Methanol (HPLC-grade or higher; numerous vendors).
49. Microcentrifuge tubes: 0.5, 1.5, and 2 mL (all nuclease-free;
numerous vendors).
50. Microcentrifuge tubes with screw caps for bead mill (optional):
2 mL (nuclease-free; e.g., MP Biomedical).
51. Microcentrifuge tube racks (plastic; numerous vendors).
52. Mixing apparatus: shaker table or dedicated mixing device
(e.g., ELMI’s Intelli-Mixer®).
53. Mortar and pestle (pre-sterilized).
54. Nitrile gloves (do not use latex or vinyl).
55. Pasteur pipette (glass and with bulb).
56. Pellet Paint-NF™ (optional; Millepore).
57. Pipets: 1–20 μL, 20–200 μL, and 200–1000 μL (e.g., Eppen-
dorf or Rainin).
58. Pipet tips-filtered only: 20, 200, and 1000 μL (do not use
non-filtered tips with this protocol).
59. Protein gel stain (e.g., Invitrogen’s SimplyBlue™ Safe Stain).
60. Protein molecular weight ladder pre-stained (numerous
vendors).
61. Protein quantification kit: BCA (numerous vendors) or Amer-
sham’s 2D-Quant™.
62. Protein wash I* (PWI): 0.3 M guanidine HCl in 95% ethanol
with 2.5% glycerol:
(a) This solution features a much lower concentration of
chaotropic salt than the BEB.
(b) The guanidine HCl should normally fully dissolve into the
ethanol in several minutes.
(c) Protect solution from light and remake fresh every few
weeks.
63. Protein wash II* (PWII): 95% ethanol with 2.5% glycerol.
64. RNAse Away® or comparable product for eliminating RNAses
from labware.
65. RNA spin column kit (optional; e.g., Qiagen).
66. Safety goggles.
67. Scalpel or razor blade (optional).
68. Sodium acetate (powder or as a 3 M solution in water
[pH 5.2]; numerous vendors).
69. Sodium chloride (NaCl; numerous vendors).
70. Sodium citrate (Na citrate; numerous vendors).
71. Sodium dodecyl sulfate (SDS; numerous vendors).
72. Sonicator bath.
73. Spatulas.
74. Spectrophotometer: Nanodrop-like unit for nucleic acids and
plate-reading unit for proteins.
75. Speed vacuum (speed vac) or lyophilizer (numerous vendors).
76. Tandem mass tags (TMT; TFS; this protocol is not discussed
herein but should be considered as an alternative to iTRAQ).
77. Triethyl ammonium bicarbonate (TEAB; e.g., TFS).
78. Trifluoroacetic acid (TFA; numerous vendors).
79. Tris-2-carboxyethyl-phosphine (TCEP; 100%; numerous
vendors).
80. Tris base (powder; numerous vendors).
81. Tris–HCl (pH 6.8): can make a higher concentration (~1 M)
and dilute as necessary.
82. Tris–borate–EDTA (TBE; purchase as concentrated stock
[e.g., 10×] or make in-house).
83. Tris–EDTA (TE; purchase as concentrated stock or make
in-house).
84. TRIzol (TFS; similar products [e.g., TRI-Reagent™] can be
substituted, but DNA quality may suffer).
85. Trypsin (sequencing grade [aka “modified”]; e.g., Promega;
cat. V5111).
86. Tubing for inert gas tanks (optional).
87. Urea (powder; numerous vendors).
88. Vortex Genie™ or comparable (numerous vendors).
Another random document with
no related content on Scribd:
paraguas con cuerda de guitarra.
Y aquí debo advertir, para lo que queda dicho y lo que siga, que
aunque no siempre poseíamos el material necesario para construir
un juguete, le adquiríamos en la plaza por medio del cambio. Había,
por ejemplo, quien estaba sobrado de astillas de pino, y no tenía
una triste tachuela: se iba á buscar con paciencia al de las
tachuelas, y se le proponían astillas en pago, ó botones, ó canicas,
ó lo que tuviéramos ó pudiéramos adquirir complicando el
procedimiento.
La pelota.—Las de orillo solo no botaban: se necesitaba goma con
él; pero la goma era muy rara en la plaza: no había otro remedio,
por lo común, que acopiar tirantes viejos y sacar de ellos, y de los
propios en uso, los hilos de goma que tuvieran, hacer una bola con
éstos, mascarla después durante algunas horas, envolverla en
cintos con mucho cuidado, y dar al envoltorio resultante unas
puntadas que unieran todas las orillas sueltas. Así la pelota, había
que forrarla. Primero se buscaba la badana por el método ya
explicado, si no nos la proporcionaba algún sombrero viejo; después
se cortaban las dos tiras, operación dificilísima que pocos
muchachos sabían ejecutar sin patrón, y, por último, se cosían con
cabo, pero poniendo sumo cuidado en no dejar pliegues ni
costurones que pudieran ser causa de que, al probar la pelota, en
vez de dar ésta el bote derecho, tomara la oblicua, lo cual era como
no tener pelota.
El látigo.—No conocí ninguno hecho de una sola cuerda nueva:
todos eran de pedazos heterogéneos, rebañados aquí y allá; pero á
dar estallido seco y penetrante, podían apostárselas con los más
primorosos: para eso se untaba muy á menudo la tralla con pez ó
con cera.
Y el taco.—Era de saúco, y saúco bueno no le había más que en
Cajo ó en Pronillo; y no en bardales públicos, sino en cercados de
huertas muy estimadas. Cuestión de medio día para cortarle, y
capítulo, por ende, de correr la clase correspondiente.
Un chico que ya había cortado allí el suyo, nos acompañaba á los
varios que le necesitábamos. El más fuerte y más ágil trepaba al
arbusto con la navaja descoyuntada, ya descrita, mientras los otros
vigilaban el terreno y le indicaban la mejor rama. Enredábase con
ella el de arriba, echando maldiciones á la navaja, que tanto le
pellizcaba el pellejo de la diestra entre la hoja y el muelle trasero,
como penetraba en el saúco.
Al cabo de media hora, cuando la rama empezaba á ceder y la
mano á sangrar á chorros, aparecía el amo á lo lejos.—«¡Ah,
pícaro!... ¡ah, tunante!... ¡yo te daré saúco en las costillas!» El de la
rama hacía un esfuerzo supremo; arrancábala, más bien que la
cortaba, y se arrojaba con ella al suelo, quedando en él medio
despanzurrado. Alzábase en seguida por amor al pellejo; y corre
que te corre con sus camaradas, no parábamos hasta los Cuatro
Caminos.
Allí nos creíamos seguros y nos poníamos á examinar el botín de la
campaña.
—¡Es hembra!—decía uno al instante.
—¡Hembra es!—exclamábamos luego los demás, tristes y
desalentados.
Trabajo perdido. Llámase saúco hembra al que tiene mucho pan, ó
médula; y el taco, para ser bueno, ha de ser de saúco macho, es
decir, de poco pan.
Vuelta á empezar en Cajo, si el sobresalto fué en Pronillo, ó en
Pronillo, si el susto le recibimos en Cajo.
Obtenido al cabo el buen saúco, á costa de trabajos como el citado,
se cortaba en porciones adecuadas; se le sacaba el pan y se
despellejaba. Esto se hacía por el camino volviendo á casa, cargada
la conciencia con el peso de la clase corrida.
Ya teníamos taco; pero se necesitaban balas y baqueta.
Las primeras eran de estopa; y como no la había á la vista,
recurríamos á las entretelas de la chaqueta, donde abundaba
siempre, merced al rumbo de los sastres de entonces; ó al plato de
la gorra, que también lo tenía por mullida. Áspera era y mala, y
plegada de inmundicias estaba; pero, al cabo, era estopa, y llegaba
á servirnos después de macerarla en la boca con paciencia y sin
escrúpulos. La baqueta era de alambre gordo, con mango de palo;
las cuales materias se adquirían como la necesidad nos daba á
entender, y nunca tan pronto como deseábamos.
Este juguete era uno de los que más nos entretenían, no sé si por
los sudores que nos costaba; y aunque con la boca del estómago
dolorida de apoyar en ella el mango de la baqueta, y las palmas de
las manos henchidas de recibir las balas al salir del taco con el
estruendo apetecido, y las fauces secas por haber gastado la saliva
en remojar las balas, siempre nos daba pena ver acabarse el tiempo
de los tacos y empezar el otro juego que, por sucesión inalterada,
estaba llamado á reinar entre los muchachos.
He puesto con el taco fin á la lista de juguetes de mis tiempos, por
no hacerla interminable, y porque bastan los descritos para dar una
idea de los sudores que nos costaba adquirir el más insignificante
de ellos, y, por tanto, del aprecio en que los tendríamos. Si algún día
me encuentro con el humor necesario, hablaré de los restantes, y
hasta de cierta corrida de toros artificiales que dimos, siendo yo
coempresario de ella, en un corralón de la calle de Cervantes, á la
cual acudió tanta gente, que, siendo á dos cuartos la entrada,
después de cubrir todos los gastos le quedaron horros á la empresa
doce reales y pico.
Entre tanto, vea el lector desapasionado si de las estrecheces y
apreturas de aquellos tiempos se deduce alguna ventaja
transcendental. De mí, le diré que en víspera de estreno de vestido,
nunca dormí sueño sosegado, y que jamás he olido perfume que me
embriague como el hedor del betún de los borceguíes recién traídos
de la zapatería, y, de propio intento, puestos por mí debajo de la
cama el sábado por la noche... Digo mal: otro olor de aquellos
tiempos me impresionó más todavía que el de los borceguíes: el olor
del teatro la primera vez que me dió en las narices, un domingo por
la tarde. Fuí solo; y cuando entré, comenzaba á bajar la araña por el
agujero de la techumbre, encendidos sus mecheros de aceite; y
según iba bajando, iba yo, á su luz, orientándome en aquél, poco
antes y aún mucho después, misterio conmovedor. Vi el telón de
boca, con las nueve Musas y Apolo pintados en él. De pronto creí
que aquellas figuras eran toda la función, y casi me daba por
satisfecho; ó que si algún personaje más se necesitaba, aparecería
entre el telón y las candilejas, y entonces me sentía hasta
reconocido, y aun hallaba muy holgado el terreno en que, á mi
entender, habían de moverse. Después sonó la música: la polka
primitiva y el himno de Vargas. ¡Qué sorpresa, Dios mío! Por último,
se alzó el telón: ¡qué maravillas en el escenario!... y empezó la
representación de El hombre de la Selva Negra. Con decir que me
faltó poco para ir al despacho de billetes á preguntar si se habían
equivocado al llevarme tan poco dinero por tanta felicidad, digo lo
que sentí en tan supremos instantes, y cuán por lo serio tomé lo que
en el escenario sucedía.
Por eso no se escandalice nadie si me oye decir alguna vez que los
actores que pone mi corazón sobre todos los del mundo conocido,
son Fuentes, la Fenoquio y Perico García, galán, dama y gracioso,
respectivamente, que trabajaron en aquella función memorable y en
otras á que logré asistir después. Pues todos estos recuerdos y las
subsiguientes emociones me asaltan y acometen siempre que á mi
olfato llega el olor de teatro vacío como estaba, ó poco menos, el de
Santander, cuando en él entré por vez primera.
Apuntados estos detalles, que fácilmente dan la medida de otros mil
del propio tiempo, recuerden mis coetáneos qué idea se tenía, entre
las gentes, de ciertos casos y de ciertas cosas. ¡Un ministro!... Boca
abajo todo el mundo. ¡Un diputado!... ¡Uff! no cabía en la calle. ¡El
Jefe político!... ¡María Santísima!... ¡Un particular que había estado
en París!... ¡Qué admiración!
En cambio, quien tenga hoy un hijo rapazuelo, que le pregunte
adónde ha ido á parar el primoroso juguete que se le compró tres
días antes, y cómo era. Ya no se acuerda de lo uno ni de lo otro. ¡Le
regalan tantos cada semana! ¡Hay tal abundancia y tal variedad de
ellos en esas tiendas de Dios!... Pregúntele también qué siente
cuando estrena un vestido ó va al teatro... Absolutamente nada.
¡Qué ha de sentir si cada día le ponen uno diferente, y concurre al
teatro todos los domingos desde que aún no sabía hablar?
Ofrézcale llevarle á Madrid dentro de un año, si saca buena nota en
los exámenes de la escuela. ¡Qué efecto ha de causarle la promesa,
si ya ha estado tres veces con su mamá en París, una para
arreglarse los dientes, otra para que le redujeran una hernia, y otra
de paso para Alemania á curarse las lombrices?
Pues salgan ustedes á la calle y pronuncien muy recio las palabras
«ministro», «diputado» y «gobernador:» las cuatro quintas partes de
los transeúntes vuelven la cabeza, porque los unos le han sido ya, y
los otros aspiran á serlo.
Ahora bien: si es preferible esta aridez del espíritu, esta dureza
precoz del sentimiento, como producto necesario del torbellino de
ideas, de sucesos y de aspiraciones en que, lustros ha, nos
agitamos, á aquellos apacibles tiempos en los cuales se dormían en
nosotros los deseos, y era la memoria virgen tabla en que todo se
esculpía para no borrarse nunca, dígalo quien entienda un poco de
achaques de la vida.
Pero conste, en apoyo de mi tesis, que hubo un día, que yo
recuerdo (y cuenta que aún no soy viejo) en que la familia española,
impulsada por el reflujo de vecinas tempestades, pasó de un salto
desde la patriarcal parsimonia de que dan una idea los pormenores
apuntados, á este otro mundo en que la existencia parece un viaje
en ferrocarril, durante el cual todo se recorre y nada se graba en la
mente ni en el corazón; viaje sin tregua ni respiro, como si aún nos
pareciera largo el breve sendero que nos conduce al término fatal,
donde han de confundirse en un solo puñado de tierra todos los
afanes de los viajeros, todas las ambiciones y todas las pompas y
vanidades humanas.
1877.
NOTAS:
[6] N. del A. en diciembre de 1880: D. José Sáenz de Miera,
milagrosamente salvado de la catástrofe del Ebro, al atravesarle
en Logroño, el 1.° de septiembre de este año, parte del
regimiento de Valencia, cuyo coronel era á la sazón.
N. del A. en octubre de 1898: Es general de división tiempo hace;
y aunque llegue á capitán general, no creeré yo que ha
ascendido todo lo que merece.
MÁS REMINISCENCIAS
Creo que fué Mad. de Sévigné quien dijo que sucede con los
recuerdos lo que con las cerezas en canastilla: se tira de una y
salen muchas enredadas. Los evocados en el cuadro anterior traen
á mi memoria otros mil, íntima y necesariamente encadenados á
ellos.
El primero que me asalta es el de mi ingreso en el Instituto
Cántabro; suceso por todo extremo señalado en aquella edad y en
aquellos tiempos, y muy especialmente para mí, y otros pocos más,
por las singularísimas circunstancias que concurrieron en el paso,
verdaderamente heroico y transcendental.
Comúnmente, pasar de la escuela de primeras letras al Instituto de
segunda enseñanza, era, y es, cambiar de local, de maestro y de
libros, y ascender un grado en categoría. El trabajo viene á ser el
mismo en una y otra región, y aun menos engorroso y molesto en la
segunda. Pero entrar en el Instituto el año en que yo entré, saliendo,
como yo había salido, de la escuela de Rojí, donde le trataban á uno
hasta con mimos, era como dejar el blando y regalado lecho en que
se ha soñado con la gloria celestial, para ponerse delante de un toro
del Jarama, ó meterse, desnudo é indefenso, en la jaula de un oso
blanco en ayunas.
Fué aquel año el último en que rigió el antiguo plan de enseñanza,
es decir, que la segunda se comenzaba por el latín solo, y que aún
conservaba esta asignatura sus tradicionales categorías de
menores, medianos y mayores. Enseñaba á los minoristas el
catedrático don Santiago de Córdoba, trasmerano inofensivo, tan
laborioso y emprendedor como desgraciado en sus empresas hasta
el fin de su vida. Este buen señor, á quien estimaban mucho sus
discípulos por la sencillez bondadosa de su carácter, hallábase á la
sazón viajando, creo que por motivos de salud, y estaba
encomendada su cátedra, interinamente, al propietario de la de
medianos y mayores, con el Ítem más de la Retórica y Poética, el
campurriano archifamoso, perínclito é inolvidable don Bernabé
Sáinz; ejemplo vivo de dómines sin entrañas, espanto y
consternación de los incipientes humanistas santanderinos de
entonces, y de muchos años antes y de algunos después.
Pasar de don Santiago á don Bernabé, no dejaba de ser duro y
penoso; pero, al cabo, había cierta semejanza entre uno y otro
territorio: los dos eran fronterizos en el edificio del Instituto, y los
moradores del primero tenían ya hecho el oído y el corazón á los
alaridos que á cada instante partían del segundo, entre el zumbar de
la vara y el crujir de los bancos derrengados; el espíritu se
empapaba en la idea de aquellos tormentos inevitables, y siquiera,
siquiera, cuando era llegado el día de comenzar á padecerlos, se
llevaban vencidos ya los riesgos de la aclimatación. Pero yo, Dios
mío, que no conocía de aquellos horrores más que la fama, y,
candoroso borrego, iba á pasar de un salto desde el apacible redil á
la caverna del lobo, ¡qué no sentiría en mis carnes, vírgenes de todo
verdascazo, y en mi corazón, formado en el amor de la familia y
entre las insípidas emociones de la escuela que acababa de dejar?
Dígaseme ahora si exagero al decir que en aquel paso memorable
había heroísmo y transcendencia.
Le di un día, ya comenzado el curso, acompañado de mi padre.
Después de haber estado en la secretaría á pagar la matrícula y
arreglar el indispensable expedienteo, me entregaron un papelejo,
especie de filiación, y con él en la mano, y temblándome las piernas,
por orden del secretario bajamos á la cátedra de don Bernabé. La tal
cátedra, como todas las de entonces, se reducía á un salón con una
puerta y algunas ventanas; un largo banco de pino, bajo y angosto,
arrimado á cada pared lateral; en la testera, y entre los dos bancos,
una mesa con tapete verde y tintero de estaño, y enfrente la puerta.
Cuando por ella entramos mi padre y yo, los bancos estaban llenos
de muchachos tristes y encogidos, y don Bernabé sentado á la
mesa. Levantáronse á una, al vernos, los infelices, que me
parecieron bestezuelas destinadas al sacrificio sobre el dolmen de
aquel druida fanático, y también se levantó éste, no por mí, sino por
mi padre. Me atreví á mirarle mientras contestaba al saludo
reverentísimo que le habíamos hecho. Me espantó aquella mueca
que en él se llamaba sonrisa. Era de mediana estatura, tenía la
frente angosta, bastante pelo, ojos pequeñitos, boca grande, labios
apretados, pómulos salientes, largas orejas, color pálido, rugoso el
cutis y muy afeitada la barba. Todo esto ocupaba poquísimo terreno,
porque la cabeza era pequeña. El pescuezo y las quijadas se
escondían entre los foques de un navajero muy planchado y una
corbata negra de armadura, con el nudo atrás, oculto por el cuello
de la levita, ó gabán, pues de ambas prendas tenía algo la que él
usaba. Seguía á la corbata, en sentido descendente, un chaleco de
terciopelo negro, abierto de solapas, entre las que se veían los
ramales, unidos por un pasador, de un grueso cordón de seda, que
iban á parar, como supe andando el tiempo, al bolsillo de la cintura
del pantalón. Éste era de paño verdoso, con trabillas postizas que
pasaban por debajo de unos pies con los juanetes más tremendos
que yo he visto. Era de notar que los zapatos de aquellos pies
semejaban ébano bruñido; la camisa era blanca y tersa, y en la ropa
no descubriría una mancha el microscopio, ni un cañón de barba en
aquella cara de marfil arranciado.
Cuando le entregamos el papelejo, nos le devolvió después de
leerle.
—¿No apunta usted su nombre?—le preguntó mi padre.
—No hay para qué—respondió.—No se me olvidará.
Tampoco á mí se me han olvidado jamás aquellas palabras, ni la
mueca con que las acompañó al mirarme de hito en hito.
Bajé yo los ojos, como cuando relampaguea, y clavé la vista en la
mesa. Había en ella, junto al tintero, un atril chiquito con dos balas
de plomo al extremo de un cordelillo cada una, con las cuales
sujetaba las hojas de un libro abierto. Á la derecha de este atril, un
bastón de ballena con puño y regatón de plata, y á la izquierda un
manojo de varas de distintos gruesos, longitudes y maderas. De
aquel bastón y de aquellas varas, ó de otras parecidas, habían
llegado á mis oídos sangrientas historias. Así es que el no se me
olvidará, juntamente con este cuadro de tortura, hizo en todo mi
organismo una impresión indescriptible.
Aún no había leído yo la Odisea, ni sabía que tal poema existiese en
el mundo, y, sin embargo, presentí, adiviné las mortales angustias
de los compañeros de Ulises en la caverna de Polifemo, al ver á
este monstruo zamparse á dos de ellos para hacer boca.
Díjonos, no el Cíclope, sino don Bernabé, qué libros necesitaba yo y
dónde se vendían; qué horas había de cátedra por la mañana y por
la tarde; encargóme que no faltara al día siguiente, y á mi padre que
no me diera mimos, porque «aquello no era cosa de juego», y volví
á ver la luz del mundo; pero ¡ay! con los ojos con que debe verla el
reo que deja la cárcel para subir al patíbulo.
Notó mi padre algo de lo que por mí pasaba, y díjome por vía de
consuelo:
—No te apesadumbres, hijo; que si, andando los días, resulta que
es tan bárbaro como cuentan, con no volver á verle está el asunto
despachado.
¡Cuánto agradeció á mi padre aquel auxilio mi espíritu angustiado!
II
La primera noticia que me dieron los minoristas antes de entrar en
cátedra al día siguiente, fué que don Bernabé no podía verlos, más
que por minoristas, por ser para él carga pesada y hacienda del
vecino. Y yo dije para mí: si ese señor desloma á los discípulos á
quienes ama por suyos, ¿qué hará con los ajenos cuando dice que
no los puede ver?
¡Ay! no me engañaban mis conjeturas ni mis condiscípulos, ni la
fama mentía. El frío de la muerte, la lobreguez de los calabozos, el
hedor de los antros, el desconsuelo, el dolor y las tristezas del
suplicio se respiraban en aquella cátedra. La vara, el bastón, la
tabaquera, el atril de las balas, la impasible faz del dómine, sus
zumbas sangrientas, su voz destemplada y penetrante como puñal
de acero, apenas descansaban un punto. Mal lo pasaban los
novicios; pero no lo pasaban mucho mejor los padres graves: para
todos había palos, y pellizcos, y sopapos, y denuestos.
Virgilio y Dante, tan diestros en pintar infiernos y torturas, se vieran
en grave apuro al trazar aquellos cuadros que no se borrarán de mi
memoria en todos los días de mi vida; porque es de saberse que,
con ser grande en aquel infierno la pena de sentido, la superaba en
mucho la de daño. Juzgábame yo allí fuera del amparo de la familia
y de la protección de las leyes del Estado; oía zumbar la vara y los
quejidos de las víctimas, y las lecciones eran muchas y largas, y no
se admitían excusas para no saberlas; y no saberlas, era tener
encima el palo y la burla, que también dolía, y la caja de rapé, y el
atril de las balas, y el calabozo, y los mojicones, y el truculento
azote, y la ignominia de todas estas cosas. ¡Quién es el guapo que
pintara con verdad escenas tales, si lo más terrible de ellas era lo
que sentía el espíritu y no lo que veían los ojos ni lo que padecía la
carne!
Lo diario, lo infalible, venía por el siguiente camino: Como no todos
los condiscípulos dábamos una misma lección, cada uno de
nosotros tenía un tomador de ella, impuesto por el dómine, el cual
tomador era, á su vez, tomado por otro. Hay que advertir también
que cuatro puntos eran malè, entendiéndose por punto la más leve
equivocación de palabra. Había de saberse la lección materialmente
al pie de la letra; de modo que bastaba decir pero en lugar de más,
para que el tomador hiciera una raya con la uña en el margen del
libro, después de las tres acometidas que se permitían en las dudas
ó en los tropezones. Dadas las lecciones, don Bernabé, desde su
asiento, iba preguntando en alta voz, por el orden en que estábamos
colocados en los bancos:
—¿Fulano?
—Dos,—respondía, por ejemplo, su tomador.
—¿Mengano?
—Benè,—si no había echado éste ningún punto, ó
—Malè,—si había pasado de tres.
—¿Cuántos?—añadía entonces el tigre, como si se replegara ya
para dar el salto. Lo que aguardaba al infeliz, era proporcionado al
número que pronunciaran los ya convulsos labios del tomador, que,
de ordinario, y por un refinamiento de crueldad del dómine, era su
mejor amigo. Hasta seis puntos se toleraban allí sin otros castigos
para la víctima que los ya conocidos y usuales; pero en pasando de
seis, en llegando, por ejemplo, á diez, no había modo de calcular las
barbaridades que podían ocurrírsele al tirano.
Estas primeras carnicerías tenían lugar después de pasada la lista á
todo el rebaño.
Como al lector, por haberle yo dicho que solían ser grandes amigos
el tomador y el tomado, puede haberle asaltado la humanitaria idea
del perdón de los puntos en obsequio á la amistad, debo decirle que
estos perdones... digo mal (pues nadie fué tan valiente que á tanto
se atreviera), las apariencias de ellos, dejaron rastros de sangre en
aquel infierno en que se castiga el perdón como el mayor de los
pecados. Apariencias de él había siempre, y no otra cosa, en las
emboscadas del dómine, porque bastábale á éste el intento de
tomar una lección sospechosa, para que se le olvidara al que iba á
repetirla hasta el nombre que tenía.
En la traducción, en las oraciones, en el repaso, en cada estación,
en fin, de aquel diurno calvario, había las correspondientes espinas,
lanzadas y bofetones.
Todo esto, en rigor de verdad, pudiera pintársele al lector con
bastante buen colorido, y aun puede imaginárselo fácilmente sin que
yo se lo pinte con todos sus pelos y señales. Pero, en mi concepto,
no era ello lo más angustioso de aquellos inevitables trances.
Temibles son los rayos, y dolorosos, y aun mortales, sus efectos;
pero los rayos son hijos de las tormentas, y las tormentas se
anuncian y pasan; y con ciertas precauciones y esta esperanza
fundadísima, el espíritu se habitúa á contemplarlas impávido. Lo
tremendo fuera para los hombres el que las centellas dieran en la
gracia de caer también á cielo sereno, y á todas horas y en todas
partes, sin avisos ni preludios de ningún género.
¡Qué vida sería la nuestra! Pues ésa era la mía en aquella cátedra,
porque allí caían los rayos sin una sola nube. Don Bernabé no se
enfurecía jamás, ni gritaba una vez más que otra; antes parecía más
risueño y melifluo, y hasta era zumbón cuando más hacía llorar.
Hería con el palo y con la palabra, como hiere el carnicero: á sangre
fría, ejerciendo su oficio. No había modo de prevenirse contra sus
rayos, ni esperanza de que la tormenta pasase, ni, por ende,
instante seguro ni con sosiego. Ésta era la pena de daño á que yo
aludí más atrás; pena mil veces más angustiosa que la de sentido,
con ser ésta tan sangrienta.
Andando los meses, y en fuerza de estudiar aquella naturaleza
excepcional, pude descubrir en ella, como síntoma de grandes é
inmediatos sacudimientos, cierta lividez de semblante y mucha
ansia de rapé. Siempre que se presentaba en cátedra con estos
celajes, y después del despacho ordinario, ó sea la toma de
lecciones, con las subsiguientes zurribandas, mandaba echar la
traducción á alguno de los cuatro ó seis muchachos de palo seguro
que había en clase; media docenita de torpes que guardaba él como
oro en paño, para darse un desahogo á sus anchas cuando el
cuerpo se le pedía.
Sin más que oirse nombrar la víctima, como ya tenía la conciencia
de su destino, levantábase temblando; y con las rodillas encogidas y
muy metido el libro por los ojos, comenzaba á leer mal lo que había
de traducir... peor. Don Bernabé, con la cabeza gacha, la mirada
torcida, las manos cruzadas sobre los riñones, en la una el bastón
de ballena y en la otra la caja del rapé, paseábase á lo largo del
salón. Poco á poco iba acercando la línea que recorría al banco del
sentenciado. Dejábale decir sin replicarle ni corregirle. De pronto
exclamaba, con su voz dilacerante envuelta en una caricatura de
sonrisa:—¡Anda, candonga! Esta salida equivalía á un tiro en el
pecho. El pobre chico se quedaba instantáneamente sin voz, sin
sangre y con los pelos de punta.
—¡Siga, calabaza!—gritábale á poco el dómine, acercándose más al
banco.
Y seguía el muchacho, pero como sigue el reo al agonizante, seguro
de llegar muy pronto al banquillo fatal. Y el lobo siempre
acercándose, hasta que, al fin, se paraba muy pegadito á la oveja.
En esta postura, tomaba un polvo, agachaba la cabeza, arrimaba la
oreja derecha al libro y requería con la zurda el bastón. El traductor
perdía entonces hasta la vista, caían de su boca los disparates á
borbotones, y empezaba el suplicio. El primer golpe, con la caja de
rapé, era á la cabeza; el segundo, con el libro, empujado con el
puño del bastón, en las narices, al bajarlas el infeliz hacia las hojas
huyendo de los golpes de arriba; después, lanzadas, con el propio
puño de plata, al costado, al estómago, á la barriga y á los dientes;
doblábase la víctima por la cintura, y un bastonazo en las nalgas la
enderezaba; gemía con la fuerza del dolor, y un sopapo le tapaba el
resuello.
—¡Conque pasce capellas significa paz en las capillas?—decía, en
tanto, el verdugo con espantosa serenidad.—¡Candonga! eso se lo
habrán enseñado ahí enfrente.
«Ahí enfrente» era la cátedra de Córdoba.
Cuando la víctima, rendida por el dolor y por el espanto, no daba ya
juego, es decir, se sentaba resuelta á dejarse matar allí, el verdugo,
señalando una nueva con la mano, decía:
—Á ver, ¡el otro!
Y el otro seguía la interrumpida traducción; y como era de necesidad
que no anduviera más acertado que su camarada, seguían también
los palos y los denuestos. En ocasiones no lo hacían mejor los
suyos, fuera por ignorancia, fuera por espanto, al llegar á ellos el
punto dificultoso; ¡y allí eran de ver en juego todos los rayos de
aquel Júpiter inclemente! Entonces tiraba al montón, y recorría los
bancos de extremo á extremo, y hería y machacaba hasta romper
las varas en piernas, manos, espaldas, entre las orejas, y en el
pescuezo, y en el cogote; y cuando ya no tenía varas que romper,
iba á la mesa, agarraba el atril de las balas y se le arrojaba al
primero en quien se fijaran sus ojuelos fosforescentes.
Por tales rumbos solía venir lo que, no por ser extraordinario, dejaba
de ser frecuentísimo, y, sobre todo, imprevisto é inevitable.
Inevitable, puesto que ni la falta de salud, ni la pena de un luto
reciente, ni las condiciones de temperamento, ni siquiera la
incapacidad natural, valían allí por causas atenuantes. Saber ó no
saber; mejor dicho, responder ó no responder. Ésta era la ley, y allá
va un caso de prueba.
Aquel famoso don Lorenzo el Pegote, cura loco, que al fin acabó
recogido en la Casa de Caridad, tuvo el mal acuerdo de que
aprendiera latín un sobrino suyo, marinero de la calle Alta. Este
infeliz muchacho entró en el Instituto ocho días después que yo, y
sentóse á mi lado, con su elástica de bayeta amarilla, sus calzones
pardos, sus zapatones de becerro, y oliendo á parrocha que
tumbaba.
Cómo le entraría el latín á este alumno que apenas sabía deletrear
el castellano, y dejaba el achicador y las faenas de la lancha para
coger en las manos el Carrillo, júzguelo el pío lector. Ni en su
cabeza cabían las más sencillas declinaciones, ni siquiera la idea de
que pudieran servirle en los días de su vida para maldita de Dios la
cosa. En cátedra estaba siempre triste y como azorado.
Cualquier mortal de buena entraña, teniendo esto en cuenta, le
hubiera guardado muchas consideraciones; pero don Bernabé no
vió en el sobrino del loco don Lorenzo más que un discípulo que no
tragaba el latín, y desde el segundo día de conocerle comenzó á
atormentarle, con una perseverancia verdaderamente espantosa.
Preguntábale sin cesar sabiendo que había de obtener un desatino
por respuesta; y como si este desatino fuera un mordisco de fiera
acorralada, como á tal contundía y golpeaba al pobre chico, y hasta
le ensangrentaba á varazos las encallecidas palmas de las manos.
Sufríalo todo el desventurado con la resignación de un mártir, y
solamente algunas lágrimas silenciosas delataban su dolor y su
vergüenza. Tomaba el dómine la resignación por resistencia
provocativa, y entonces caía en una de aquellas borracheras de
barbarie que tan famoso hicieron su nombre en el Instituto Cántabro.
Hay que advertir que el tal don Lorenzo iba casi todos los días, con
su raído levitón, su bastón nudoso y su faz airada y fulgurante, á
preguntar á don Bernabé por los adelantos de su sobrino. Hartábase
el dómine entonces de ponderarle la torpeza y holgazanería del
muchacho; con lo cual aquel energúmeno, que tenía por costumbre
apalear en la calle á cuantos le miraban con cierta atención,
deslomaba al sobrino en cuanto éste volvía á casa, harto más
necesitado de bizmas y buen caldo.
Antes de cumplirse un mes, ahorcó los libros el callealtero, y se
metió á pescador, en el cual oficio se había amamantado. Y fué un
sabio acuerdo. La vida que traía de estudiante no era para tirar más
allá de cinco semanas con pellejo. Desde entonces no he vuelto á
verle, ni he sabido nada de él. Pero dudo que, por perra que haya
sido su suerte, envidiara la que tuvo al caer bajo la férula sangrienta
de don Bernabé Sáinz.
Qué carnes y qué corazón se me pondrían á mí contemplando estos
cotidianos espectáculos, júzguelo el pío lector. Yo no comía, yo no
sosegaba. Como don Quijote con los libros de Caballerías, me
pasaba las noches de claro en claro, estudiando el Carrillo, sacando
oraciones y traduciendo á Orodea; y con tal ansia devoré aquel Arte,
tan á mazo y escoplo le grabé en la memoria, que hoy, al cabo de
treinta y más años, me comprometería á relatarle, después de una
sencilla lectura, sin errar punto ni coma. Y no obstante, más que á
estas faenas sobrehumanas, atribuyo á la Providencia el milagro de
no haber probado de las iras de don Bernabé sino lo que me tocó en
los vapuleos generales, que nunca fué mucho. ¡Pero qué
inquietudes! ¡Pero qué temores! ¡Pero qué pesadillas!... vuelvo á
decir. Desde entonces niego yo que pueda nadie enfermar, y mucho
menos morir, de susto, puesto que sobreviví á tal cúmulo de
horrores, tan extraños é inconcebibles en mi edad y especiales
condiciones de temperamento.
Algunas veces, al presenciar una de aquellas matanzas, atrevíame
á preguntarme con el pensamiento, pues con la lengua hubiera sido
tanto como quedarme sin ella en el gaznate: ¿Qué razón puede
haber para que faltas tan leves merezcan castigos tan horrendos?
¿Cómo hay padres que consientan que un extraño maltrate de este
modo á sus hijos? ¿Quién ha dado á este hombre tan bárbaras
atribuciones? Si esto es cátedra, sobran los palos; si matadero,
sobran los libros».
Pero como había padres que aplaudían aquellas tundas feroces
administradas á sus hijos, y leyes que no ataban las manos al
verdugo, calculaba yo que así, y no de otro modo, deberían andar
aquellas cosas.
Refiere Suetonio que hallándose Tiberio en Caprea, una tarde en
que se paseaba junto al mar, un pescador se le acercó muy quedito
por detrás y le ofreció una hermosa lobina que aún coleaba.
Sorprendido el tirano por la voz del pobre hombre, mandó que le
restregaran la cara con el pez, en castigo de su atrevimiento; y así
se hizo. «Fortuna—dijo luego el pescador, mientras se limpiaba la
sangre que corría por sus mejillas,—que no se me ocurrió ofrecerle
una langosta».
No por el dicho del pescador, sino por el hecho del tirano, saco el
cuento á relucir aquí, y esto, para que se vea que, en ocasiones,
don Bernabé daba quince y falta á Tiberio. Verbi gracia: para el
acopio de varas se valía de los mismos discípulos á quienes
vapuleaba todos los días; y hubo ocasión en que rompió sobre las
nalgas de uno de ellos todas cuantas acababa de presentarle,
recién cortadas con ímprobos trabajos, so pretexto de que no eran
de las buenas, y que además estaban picadas con el cortaplumas.
Pudiera citar muchos ejemplos de esta especie; pero cállolos,
porque no se crea que me ensaño en la memoria de este hombre,
que ¡admírese el lector! á pesar de lo relatado, no fué merecedor de
tal castigo.
III
Esta figura trágica tenía también su lado cómico en la cátedra, no
por intento del catedrático, sino por la fuerza del contraste; como es
alegre, en un cielo negro y preñado de tempestades, la luz del
relámpago que en ocasiones mata.
Don Bernabé era hombre de cortísimos alcances, y esto se echaba
de ver en cuanto se salía de los carriles del Arte señalado de texto.
En tales casos, hubiera sido, como ahora se dice, verdaderamente
delicioso, por la especialidad de los recursos que le suministraba la
angostura de su ingenio, si su lado cómico no hubiera estado tan
cerca del dramático.
Fuera de los poquísimos ejemplos que sacaba de Tácito ó de Tito
Livio, todas sus oraciones, propuestas muy lentamente y
paseándose á lo largo de la clase, tomando rapé, eran del género
culinario, y, mutatis mutandis, siempre las mismas.
—Estando la cocinera—díjome una vez, al llegar yo á hacer
oraciones de esta clase,—fregando los platos, los discípulos le
robaban el chocolate.
Puse en latín, y sin grandes dificultades, lo de la cocinera, lo de
fregar los platos y hasta lo del robo por los discípulos; pero llegué al
chocolate y detúveme.
—¿Qué hay por chocolate?—pregunté.
—Hombre—me respondió deteniéndose él también en su paseo,
torciendo la cabecita y tomando otro polvo,—la verdad es que los
romanos no le conocieron.—Meditó unos momentos, y añadió, con
aquella voz destemplada, verdadera salida de tono, que le era
peculiar:
—Ponga masa cum cacao, cum sácaro et cum cinamomo confecta.
(Masa hecha con cacao, azúcar y canela).
Hízonos gracia la retahila, y reímonos todos; pero pudo haberme
costado lágrimas la dificultad en que me vi para acomodar tantas
cosas en sustitución del sencillísimo chocolate, sin faltar á la ley de
las concordancias en genero, número y caso.
Otra vez, y también á propósito de fregonas y de discípulos golosos,
salió á colación la palabra arroba.
—¿Qué hay por arroba?—preguntó el alumno.
Á lo que respondió don Bernabé, con la voz y los preparativos de
costumbre:
—La verdad es, candonga, que esa unidad no la conocían los
romanos... Ponga... pondus viginti et quinque librarum (peso de
veinticinco libras).
Como en estos casos le daba por estirarse, en otros prefería
encogerse.
—Voy á Carriedo,—mandó poner en latín en una ocasión; y como el
alumno vacilase,
—¡Carretum eo, candonga!—concluyó el dómine alumbrándole dos
estacazos.—¿Qué ha de haber por Carriedo sino Carretum, carreti?
Cuando un muchacho quería salir de la cátedra, obligado á ello por
alguna necesidad apremiante, ó fingiendo que la sentía, alzábase
del banco, cruzaba los brazos sobre el pecho, y quedábase mirando
á don Bernabé, con la cara muy compungida; hacía éste un
movimiento expresivo con la cabeza, y así concedía ó negaba el
permiso que se le pedía.

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Lipidomics: Methods and Protocols 2nd

Edition Sanjoy K. Bhattacharya (Editor)

NEURONAL CELL CULTURE methods and protocols 2nd Edition

Ovarian Cancer Methods and Protocols Methods in

Antibiotics Methods and Protocols 2nd Edition Peter

Peroxisomes Methods and Protocols 2nd edition Michael

Chaperones: Methods and Protocols 2nd Edition John M.

MicroRNA Profiling Methods and Protocols 2nd Edition

MicroRNA Profiling Methods and Protocols 2nd Edition

CHLOROPLAST BIOTECHNOLOGY methods and protocols 2nd

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Miami, FL, USA Sanjoy K. Bhattacharya

1 Isolation of Mitochondrial Lipids and Mass Spectrometric Analysis . . . . . . . . . . . 1

13 Analysis of Cholesterol Lipids Using Gas Chromatography

Integrative Metabolomics Research Center, Miami, FL, USA; Molecular Cellular

ROBERTO MARIA PELLEGRINO • Department of Chemistry, Biology and Biotechnology,

CHARLES YAROS • Department of Ophthalmology, University of Miami, McKnight Vision

Isolation of Mitochondrial Lipids and Mass Spectrometric

Key words Mitochondrial lipids, Neurodegeneration, Lipidomics, Liquid chromatography, mass

Mitochondria are gaining rapid popularity among translational

Sanjoy K. Bhattacharya (ed.), Lipidomics: Methods and Protocols,

mitochondrial membrane (IMM) contains approximately 20% of

Prepare all solutions using LC-MS grade solvents and chemicals to

8. Pierce Micro BCA Protein Assay Kit.

2.2 Other Materials 1. Dounce homogenizer.

2.3 High- 1. Accucore™ Vanquish™ C18+ Column

3.1 Sample 1. Dry and weigh optic nerves to approximately 1 mg.

11. Add 200 μL of Reagent C (Mitochondrial Isolation Kit for

3.2 Butanol– 1. Resuspend mitochondrial pellets in 300 μL of n-butanol–

3.3 High- 1. Resuspend dried lipid samples in 50 μL of chloroform/metha-

4. Prepare and label LC-MS vials for both positive and

3.4 Lipid 1. Upload raw data produced by LC-MS/MS to LipidSearch™.

1. This protocol was modified for use in small sample sizes. I

Isolation of Neuronal Synaptic Membranes by Sucrose

Sucrose gradient centrifugation has been used by biochemists span-

Sanjoy K. Bhattacharya (ed.), Lipidomics: Methods and Protocols,

of Redburn’s protocol to resolve these large and small synapses

identifying new lipid molecules that, like docosahexaenoic acid and

2.1 Solutions 1. 0.32 M sucrose/4 mM HEPES (300 mL): 32.86 g sucrose,

2.2 Equipment 1. Centrifuge: Beckman Coulter Optima L-80 XP

As a general rule, 1 mL of buffer is used per 15–20 mg wet weight

4. After tissue settled to the bottom of the tube, replace buffer

STOP: At this stage you have isolated your neurosynapto-

4. Resuspend final pellets in an appropriate volume of wash buffer

1. The volume unit can be adjusted if needed; lowering the tissue

Heather VanGuilder Starkey for sharing her isolation protocols

6. VanGuilder HD, Brucklacher RM, Patel K, 9. Martoft L, Stodkilde-Jorgensen H, Forslid A,

Multi-macromolecular Extraction from Endosymbiotic

1.1 Safety High-efficiency extraction of RNAs, DNAs, proteins, lipids, and

Sanjoy K. Bhattacharya (ed.), Lipidomics: Methods and Protocols,

Occupational Health and Safety Administration (OSHA). I recom-

Fig. 1 An Indo-Pacific coral reef featuring a high abundance of a branching coral

Fig. 2 A scanning electron micrograph of decalcified, freeze-fractured reef coral

starvation [11], are “background players” with respect to their

Simple-random-sample of 20 endosymbiotic anthozoan molecular biology articles

While genome size is static within a living cell, it is more

Where technique becomes more critical, though, is in the lysing

Fig. 3 A schematic featuring a brain coral (~50 cm in diameter) and a depiction

safer approach featuring commercially available spin columns

1.4 Protocol Analysis of complex suites of macromolecules—namely, RNAs,

Option A (Fig. 5) and Option B (Fig. 6). The former is superior in