Western blotting

Principle: The method is characterized by transferring the protein which run on a gel by electrophoresis on to a nitro cellulose membrane.
It is widely used analytical technique in molecular biology to detect specific proteins in a sample of tissue homogenate or extracts.

1. Western Blotting Presented By Bhupendra Regmi M sc. Med Biochemistry 2 nd Year, JSSMC Mentor Dr. Anjali Devi Professor, Dept. of Biochemistry JSSMC 02-05-2019 Western Blotting 1

2. Contents Introduction Procedure Applications Limitations Conclusion References 02-05-2019 Western Blotting 2

3. Introduction 02-05-2019 Western Blotting 3

4. Blotting Blotting is the techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. 02-05-2019 Western Blotting 4

5. Types of Blotting Blotting Southern Blotting (DNA Detection) Northern Blotting (RNA Detection) Western Blotting (Protein Detection ) 02-05-2019 Western Blotting 5

6. Western Blotting Principle : The method is characterized by transferring the protein which run on a gel by electrophoresis on to a nitro cellulose membrane. It is widely used analytical technique in molecular biology to detect specific proteins in a sample of tissue homogenate or extracts. George Stark , Harry Towbin , W. Neal Burnette and there groups are credited for developing western blotting techniques during late 1970s . 02-05-2019 Western Blotting 6

7. Procedure 02-05-2019 Western Blotting 7

8. Sample Preparation Gel Electrophoresis Protein Transfer Protein Staining Blocking Washing, Analysis and Detection 02-05-2019 Western Blotting 8 Fig: Western Blotting Procedure

9. Sample Preparation Detergent Lysis for Tissue Culture Enzymatic Digestion for bacteria, yeast and fungal cell Ultra sonication for Cell suspension Mechanical homogenization for plant and animal tissues 02-05-2019 Western Blotting 9 Fig: Sample Preparation Technique

10. Electrophoresis Native gel SDS page 02-05-2019 Western Blotting 10

11. 02-05-2019 Western Blotting 11 Sample loading into wells of electrophoretic chamber Loading buffer into empty wells Place the safety lid on the unit Plug the color coded leads into the jacks in power supply Run the gel under the appropriate condition When the tracking gel reaches the bottom of the gel, turn off the power supply Disconnect the leads and remove the safety leads and remove the safety lids Remove the gel from the electrophoretic apparatus Process the protein transfer Fig : Page Protocol

12. 02-05-2019 Western Blotting 12 Fig: SDS PAGE Process

13. Protein Transfer Order of protein transfer 02-05-2019 Western Blotting 13 Fig : Protein Transfer

14. Protein Staining All the protein in the gel should be completely eluted As protein are not directly visible in the gel, the gel must be stained Protein are usually stained by Ponceau S, stain-free techniques, Sypro Ruby, Epicocconone , Coomassie R-350, Amido Black, and Cy5. 02-05-2019 Western Blotting 14

15. Blocking The meaningful result the antibodies must bind only to the protein of interest not to the membrane. Non specific binding (NSB) of antibodies can be reduced by blocking the unoccupied site of membrane with an inert protein or non ionic detergent Blocking agent should posses greater affinity towards membrane then the antibodies Blocking Agents BSA Non Fat meal Casein Gelatin Dilute solution of Tween 20 Colloidal carbon Western Blotting 15

16. Detection During the detection process the membrane is probed for the protein interest with antibodies and links them to reporter enzyme, which drives a colorimetric or photometric signal. There are two methods for detection T wo steps detection Primary Antibody Secondary Antibody One step detection Probe Antibody 02-05-2019 Western Blotting 16

17. Washing Unbound antibodies can cause high background and poor detection Hence washing the blot removes unbound antibodies from the membrane A dilute solution of Tween 20 in TBS or PBS buffer is commonly used for washing 02-05-2019 Western Blotting 17

18. Analysis Techniques for Analysis Colorimetric Detection Chemiluminescent Radio active Detection Fluorescent Detection 02-05-2019 Western Blotting 18

19. 02-05-2019 Western Blotting 19 Fig: Techniques for Analysis

20. 02-05-2019 Western Blotting 20 GEL DOCUMENTATION SYSTEM

21. Applications Analysis of IGg fraction purified from human plasma Diagnosis of HIV by ELISA, involves western blotting technique Western blotting technique is also used to detect some form of lyme disease Confirmatory test for Hepatitis B involves western blotting technique Western blotting test is use in the analysis of bio markers such as hormone, growth factors and cytokine This technique is also employed in gene expression technique 02-05-2019 Western Blotting 21

22. Limitations Very delicate and time consuming techniques, a minute imbalance at any level of procedure can skew the result of entire process Incorrect labelling of protein can happen due to reaction of secondary antibody Causes erroneous in bands or no bands due to insufficient transfer Well trained technicians are required for this technique Primary antibody availability is crucial It is just a semi quantitative at best, only an approx. estimation and not a precise measurements of molecular weight of protein is possible. 02-05-2019 Western Blotting 22

23. Conclusion WB technique is simply a way to identify unknown protein on a polyacrylamide gel It is sometime called as protein blotting or immune blotting It is widely used analytical technique in the field of molecular biology, immunogenetics and other biochemistry disciplines A part from the limitation of WB it is more helpful nowadays hence a discovery of western blotting technique has become boon in the field of science and technology . 02-05-2019 Western Blotting 23

24. References Yang Y, Ma H. Western blotting and ELISA techniques. Researcher. 2009;1(2):67-86. Mahmood T, Yang PC. Western blot: technique, theory, and trouble shooting. North American journal of medical sciences. 2012 Sep;4(9):429 . Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. Journal of Infectious Diseases. 1993 Feb 1;167(2):392-400 . Techniques in Biochemistry by T.Devasena and G.Rajagopal . 02-05-2019 Western Blotting 24

25. THANK YOU 02-05-2019 Western Blotting 25