MOLECULAR BIOLOGY

LABORATORY TECHNIQUES

Protein
Analysis
E U G E N E J O H N F. BA LM O RE S J R .,
MD
Outline
I. Introduction

I. Protein Separation

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

II. Protein Identification

Western Blotting

Enzyme-linked Immunosorbent Assay (ELISA)

Mass Spectrometry
Introduction
Proteins in the body form the structure of cells; make up enzymes and hormones that regulate
bodily functions; transport analytes, nutrients, and drugs throughout the body; remove metabolic
waste; and provide defenses against infection.

Among the classes of molecular analytes found inside the body, proteins provide a unique
combination of broad clinical significance and accessibility.

The analysis of proteins and clinical diagnostic tests for proteins are widely accepted in medical
technology
Sodium Dodecyl
Sulphate
Polyacrylamide Gel
Electrophoresis (SDS-
PAGE)
https://youtu.be/MILiO1XnuqQ
A technique used for separating the proteins based on
their molecular weight.
Sodium dodecyl sulphate and polyacrylamide help to
eradicate the influence of structure and charge of the
proteins, and the proteins are separated based on the
length of the polypeptide chain.
 Was first coined by Dr. Burnette in 1981 after the
eponymous southern blot for DNA and consequent
coinage of the northern blot in 1977 for RNA

WB selects for an individual protein amongst a

Western Blotting potentially significant milieu via leveraging the
specificity of antigen (Ag)-antibody (Ab) binding.
https://youtu.be/qT_AqI-MKJc
 Is a labeled immunoassay that is considered the gold
standard of immunoassays. This immunological test is
Enzyme-linked very sensitive and is used to detect and quantify
Immunosorbent substances, including antibodies, antigens, proteins,
glycoproteins, and hormones. The detection of these
Assay (ELISA) products is accomplished by complexing antibodies and
antigens to produce a measurable result.
https://youtu.be/RRbuz3VQ100
ELISA testing is used to diagnose HIV infection,
pregnancy tests, and blood typing, among others.
 Is a mainstream chemical analysis technique in the twenty-
first century. It has contributed to numerous discoveries in
chemistry, physics and biochemistry. Hundreds of research
Mass laboratories scattered all over the world use MS every day to
investigate fundamental phenomena on the molecular level.
Spectrometry Commonly regarded as an instrumental technique for
https://youtu.be/JPpKL1uzE0I separation of electrically charged species in the gas phase:
the charged species (ions) are produced in the ion source (in
some cases, the ion source also assists the transfer of solid-
phase; or liquid-phase analytes into the gas phase); the gas-
phase ions subsequently are transferred into the mass
analyser, which in turn sorts the ions—in space or time—
according to the massto-charge ratios (m/z); finally, the
separated ions are detected by an ion detector in the space or
time domain.
Thank you!