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Characterization of protein
- 1. CharacteriSation of Protein By KAUSHALKUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )
- 2. C O N T E N T Introduction – Proteins – FunctionOf Protein And Their Properties Protein Isolation And Purification Methods Of Cell Lysis Steps Of Protein Characterisation: – Determination Of Protein Concentration • Biuret Reaction • Lowry (Folin-Lowry) Method • UV- Spectroscopy – Assessment Of Protein Purity • SDS -Phage • Immunoblot – Surface Charge Analysis • Isoelectro Focusing • Ion Exchange Chromatography – Size, Shape And Conformation Analysis • 2d-Electrophorasis • X-Ray Crytalliography – Protein Structure and Sequence Analysis • Edman Sequencing Conclusion References 2
- 3. INTRODUCTION • Proteins? – Amacromolecule 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. 3
- 4. Characteristics Of protein: –Size – Shape – Concentration – Purity – Charges (+v, -ve and isoelectric) – Molecular structure and conformation – Amino acid sequence – Biological activity 4
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- 6. Chromatographic Techniques ForProtein Purification 6
- 7. Protein characterization: • Thecharacterization 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. 7
- 8. STEPS OF PROTEINCHARACTERISATION: –Determination Of Protein Concentration –Assessment Of Protein Purity –Surface Charge Analysis –Size, Shape And structure AnalysiS –Protein Sequence Analysis 8
- 9. Determination Of ProteinConcentration 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. 9
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- 11. 3. UV -spectrophotometer Concentration (mg/ml) = (1.55 x A280) – (0.76 x A260) x dilution factor 11
- 12. ASSESSMENT OF PROTEINPURITY: 1. SDS - PAGE 12
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- 14. SURFACE CHARGE ANALYSIS 1.ISOELECTRIC FOCUSING: 14
- 15. SIZE, SHAPE ANDCONFORMATION ANALYSIS 1. 2D – GELELECTROPHORESIS: 15
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- 20. CONCLUSION Protein characterization involvesthe 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. 20
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