Western Blotting Technique.

In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur

1. 1   A Seminar as a part of curricular requirement for M . Pharmacy I year I Semester Presented by T. Niranjan Reddy (20L81S0109) Department of Pharmacology Under the guidance of Dr. P. Ramalingam, MPharm , Ph. D Research Director and professor of Pharmaceutical and medicinal chemistry WESTERN BLOTTING

2. Contents : Introduction Types of blotting techniques Principle of western blotting Procedure for western blotting Gel electrophoresis Protein transfer Antibody probing Protein detection Analysis and imaging Applications Limitations 2

3. Introduction : Blotting Blotting is a method of transferring proteins, DNA or RNA, on to a carrier (for example, a nitrocellulose or PVDF or nylon membrane) 3

4. Western blotting is a widely used analytical technique in molecular biology to detect specific protein in a sample of tissue homogenate or the extract It works on the principle of gel electrophoresis Proteins are separated based on their size on polyacrylamide gel Western blotting

5. 6 Western blotting is an immunoblotting technique which rely on the specificity of bonding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules In western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein The SDS PAGE (sodium dodecyl sulphate –polyacrylamide gel ) technique is a prerequisite for western blotting . Principle of western blotting

6. 7

7. 8

8. 9 The proteins of the sample are separated by using the gel electrophoresis Electrophoresis is a commonly used method for separating proteins on the basis of size, shape and charge In SDS (sodium dodecyl sulphate) electrophoresis, protein samples are separated according to their molecular weight . Gel electrophoresis

9. 10 Page protocol

10. Sample Loading 11

11. 12 On completion of the separation of proteins by polyacrylamide gel electrophoresis, the next step is to transfer the proteins from the gels to solid support membrane This solid support membrane usually made up of a chemically inert substance such as nitrocellulose or PVDF (polyvinylidene difluoride) The process of transferring proteins from a gel to a membrane while maintaining their relative positions and resolutions is known as blotting. protein transfer :

12. 13 After gel electrophoresis it may be necessary to confirm that all the proteins in the gel have been completely eluted As proteins are not directly visible in the gel, the gel must be stained Proteins are usually stained with dyes such as Coomassie blue, sliver stain or deep purple After staining a permanent record may be made by imaging the gel with the suitable instrument. Protein staining:

13. 14

14. Blocking : The membrane has the ability to bind to proteins in this case both the target and antibodies are proteins and so there could be some unwanted binding. For meaningful results, the antibodies must bind only to the protein of interest and not to the membrane Non specific binding of antibodies can be reduced by blocking the unoccupied sites of inert protein or non-ionic protein Blocking agents should possess greater affinity towards membrane than the antibodies 15

15. 16 The most commonly used blocking agents are : Bovine serum albumin (BSA) Non –fat milk Casein Gelatine Dilute solution of tween 20 Blocking agents

16. Antibody probing After blocking, the blot is incubated with one or more antibodies. This uses specific antibody to detect a localize the protein blotted to the membrane The specificity of antigen antibody permits the identification of a single protein in a complex sample The non-labelled primary antibody directed against the target protein and specific labelled secondary antibody binds to primary antibody The secondary antibody is conjugated to an enzyme that is used to indicate the location of protein Secondary antibodies can be a monoclonal or polyclonal antibodies 17

17. Washing The 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 18

18. Protein detection After the unbound probes are washed away, the western blotting is now ready for detection of probes that are labelled and bound to the protein of interest The marked antibody which is linked to a reporter enzyme which when exposed to appropriate substrate drives a colorimetric reaction and produces a colour Enzymes such as alkaline phosphatase(AP) and horse radish peroxidase (HRP) are widely used in detection of proteins

19. 20

20. 21 This is the last and major step of the western blotting technique Detection of signals using either x-ray film, scanners or a CCD, results in one or more visible proteins on the membrane image The molecular weight of protein can be estimated by comparison with marker proteins and the amount of protein can be determined and this is related to band intensity Qualitative and quantitative analysis can be done in order to verify the absence or presence of specific proteins of interest. Analysis and imaging

21. 22 Analysis of igG fractions purifies from human plasma Diagnosis of HIV by ELISA, involves the western blotting technique Western blotting technique is also used to detect some forms of Lyme disease Western blotting technique is used in definitive test for BSE, which is commonly known as mad cow disease confirmatory test for hepatitis-B involves western blotting technique This technique are employed in the gene expression studies. Application of Western Blotting

22. 23 Very delicate and time consuming process If a protein is degraded quickly, then western blotting technique wont detect it well Well trained techniques are required for this technique Primary antibody availability is crucial. Limitations of Western Blotting

23. References : Kurien BT , Scofield RH. 2006. Western blotting. Methods 38: 283–293. Jensen EC . The Basics of Western Blotting. Anat Rec. 2012 Mar 1;295(3):369–71. Antharavally BS, et al . A high-affinity reversible protein stain for Western blots.  Analytical biochemistry.  2004;329(2):276–80 24

24. 25