Characterization of protein

Introduction
Proteins
Function Of 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

1. CharacteriSation of Protein By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. CONTENT Introduction Proteins Function Of 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? 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. 3

4. Characteristics Of protein: Size Shape Concentration Purity Charges (+v, - ve and isoelectric ) Molecular structure and conformation Amino acid sequence Biological activity 4

5. PROTEIN ISOLATION AND PURIFICATION: 5

6. Chromatographic Techniques For Protein Purification 6

7. 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. 7

8. STEPS OF PROTEIN CHARACTERISATION: Determination Of Protein Concentration Assessment Of Protein Purity Surface Charge Analysis Size, Shape And structure AnalysiS Protein Sequence Analysis 8

9. Determination Of Protein Concentration   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

10. 10

11. UV - spectrophotometer Concentration (mg/ml) = (1.55 x A280) – (0.76 x A260) x dilution factor 11

12. ASSESSMENT OF PROTEIN PURITY: SDS - PAGE 12

13. IMMUNOBLOT 13

14. SURFACE CHARGE ANALYSIS ISOELECTRIC FOCUSING: 14

15. SIZE, SHAPE AND CONFORMATION ANALYSIS 2D – GELELECTROPHORESIS: 15

16. Mass spectrophotometer 16

17. 17

18. X-ray crytalliography : 18

19. PROTEIN SEQUENCING 19

20. 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.  20

21. REFERENCES http://www.orcexperts.com/experts.asp?strSearchType=all&strQuery=protein+characterization 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 Bioinformatics and functional genomics – 3 rd edition – Jonathan Pevsner (2015) 21

22. Thank Y  u ..... 22