CharacteriSation of Protein

By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )
 Introduction
– Proteins
– Function Of Protein And Their Properties
Protein Isolation And Purification
Methods Of Cell Lysis

C Steps Of Protein Characterisation:

– Determination Of Protein Concentration

O • Biuret Reaction
• Lowry (Folin-Lowry) Method
• UV- Spectroscopy
N – Assessment Of Protein Purity
• SDS -Phage
T • Immunoblot
– Surface Charge Analysis
E • Isoelectro Focusing
• Ion Exchange Chromatography
N – Size, Shape And Conformation Analysis
• 2d-Electrophorasis
T • X-Ray Crytalliography
– Protein Structure and Sequence Analysis
• Edman Sequencing
Conclusion
References

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 INTRODUCTION
• Proteins?
– A macromolecule composed of one or more polypeptide chains,
each with a characteristic sequence of amino acids linked by
peptide bond.

• The main functions of proteins in human body are:

 They serve as body building units, e.g., muscle proteins.
 They provide support and protection to various tissues, e.g.,
collagen and keratin.
 All chemical reactions in the body are catalysed by proteinaceous
enzymes, e.g., trypsin.
 They transport various molecules and ions from one organ to the
other, e.g., hemoglobin, serum albumin.
 They store and provide nutrients, e.g., milk casein, ovalbumin.
 They defend the body from harmful foreign organisms, e.g.,
immunoglobulin’s, fibrinogen.
 They help to regulate cellular or physiological activity, e.g.,
hormones, viz., insulin, GH.

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Characteristics Of protein:
– Size
– Shape
– Concentration
– Purity
– Charges (+v, -ve and isoelectric)
– Molecular structure and conformation
– Amino acid sequence
– Biological activity

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PROTEIN ISOLATION AND
PURIFICATION:

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Chromatographic Techniques For Protein
Purification

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Protein characterization:
• The characterization of protein, which can be
based on protein size, shape, and sequence
concentration, and physiochemical properties
such as isoelectric point, molecular weight,
relative hydrophobicity, and spectral analysis.

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STEPS OF PROTEIN CHARACTERISATION:

– Determination Of Protein Concentration

– Assessment Of Protein Purity
– Surface Charge Analysis
– Size, Shape And structure AnalysiS
– Protein Sequence Analysis

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 Determination Of Protein Concentration
1. Biuret test
• When peptide bonds are present in
this alkaline solution, the Cu2+ions will
form a coordination complex with
4 nitrogen atoms from peptide bonds.
• The complex of Cu2+ ions and nitrogen
atoms make the color
of CuSO4 solution changes
from blue to violet.
• The intensity of the color is proportional
to the protein concentration.

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 3. UV - spectrophotometer

Concentration (mg/ml) = (1.55 x A280) – (0.76 x A260) x dilution factor

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ASSESSMENT OF PROTEIN PURITY:
1. SDS - PAGE

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2. IMMUNOBLOT

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SURFACE CHARGE ANALYSIS
1. ISOELECTRIC FOCUSING:

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 SIZE, SHAPE AND CONFORMATION
ANALYSIS
1. 2D – GELELECTROPHORESIS:

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Mass spectrophotometer

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3. X-ray crytalliography:

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PROTEIN SEQUENCING

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 CONCLUSION
Protein characterization involves the use of experimental methods
that allow for the detection and isolation of a protein and its
purification, as well as the characterization of its structure and
function.

The analysis of the resulting protein products can be complex and

challenging. However, accurate characterization of the final protein
that was either purified from natural sources or expressed in
different cell cultures or as a part of a drug development process is
an essential step.

The complex structure and larger size of proteins, as well as the

intrinsic nature of each protein, makes the characterization of
proteins inherently more complicated than the characterization of
smaller molecules.

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 Bioinformatics and functional genomics – 3rd edition –
Jonathan Pevsner (2015)

http://www.orcexperts.com/experts.asp?strSearchType=all&strQuery=protein+characterizati
on
https://www.medicinenet.com/script/main/art.asp?articlekey=15380
http://www.biologydiscussion.com/proteins/proteins-functions-structure-properties-and-
classification/16912
https://www.sciencedirect.com/topics/medicine-and-dentistry/isoelectric-focusing
https://www.ncbi.nlm.nih.gov/books/NBK21589/
http://www.biosyn.com/tew/protein-characterization-and-purification-methods.aspx
https://www.biologicscorp.com/protein-identification-and characterization/#.WsyIbpWgfIU

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