Western blotting in Molecular Biology and Biotechnology

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1. WESTERN BLOTTING

2. Blotting techniques involve transfer of biological samples ( DNA, RNA, or proteins ) from a gel to a membrane followed by detection on the surface of the membrane. C omplexing the target with a labeled molecule for detection. Blotting Techniques

3. Types of Blotting/Hybridization Techniques Southern blotting Northern blotting Western blotting Eastern blotting

4. Western blotting The Western blotting (alternatively, protein immunoblotting ) widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. Method developed in the laboratory of George Stark at Stanford. The name Western blot was given to the technique by W. Neal Burnette in 1981. The SDS PAGE technique is a prerequisite for Western blotting .

5. SDS-PAGE Most widely used method of analyzing protein mixture qualitatively. useful for monitoring protein purification. Based on separation of proteins according to size, can also be used to determine the relative molecular mass of the proteins. PAGE separate proteins in fractions during electrophoresis process. Individual separated fractions can be examined.

6. Principle of Western blotting It uses gel electrophoresis where proteins are separated into polyacrylamide gel according to their molecular weight. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are detected using antibodies (both monoclonal and polyclonal antibodies ) from mixture of protein.

7. Steps in a Western blotting Tissue preparation Gel electrophoresis Transfer Blocking Detection Analysis

8. Procedure

9. 1. Tissue preparation Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender, homogenizer or by sonication . Western blotting is not restricted to cellular studies, bacteria, virus or environmental samples can be the source of protein . Detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Tissue preparation is often done at cold temperatures to avoid protein denaturing and degradation.

10. 2. Gel electrophoresis . The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point ( pI ), molecular weight, electric charge , or a combination of these factors. By far the most common type of gel electrophoresis employs SDS-PAGE. It is also possible to use a two-dimensional (2-D) electrophoresis. Proteins are separated according to isoelectric point in the first dimension, and according to their molecular weight in the second dimension.

11. 3. Transfer Proteins are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF ). Transfer of proteins can be achieved either by capillary blotting or by electroblotting . Capillary Blotting The gel is placed on wet stack of buffer soaked filter papers and membrane is placed on top of the gel. Buffer is then drawn through the gel by placing a stack of dry absorbent material followed by heavy weight on top of the membrane. The entire stack is placed in a buffer solution which moves up through paper by capillary action , bringing the separated proteins on to the membrane. The process is carried out o/n.

12. A quicker (a few hrs ) and more efficient method of transfer. uses an electric current to transfer proteins from the gel into the PVDF or nitrocellulose membrane. A sandwich of gel and nitrocellulose membrane is compressed in a cassette and immersed in buffer. Current is passed, which causes separated proteins to electrophorese out of gel onto the membrane. Nitrocellulose membrane with transferred protein is referred to as blot. Electroblotting

13. Protein binding is based upon hydrophobic interactions , as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probing.

14. 4. Blocking Step must be taken to prevent interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the blot in a dilute solution of protein - typically 10% Bovine serum albumin (BSA) or 5% non-fat dried milk in Tris-Buffered Saline (TBS), with a minute percentage of detergent such as Tween 20 or Triton X-100. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. This reduces "noise" in the final product of the Western blot, leading to clearer results, and eliminates false positives.

15. 5. Detection Chemiluminescent or colorimetric detection: peroxidase

16. The enzyme used in enzyme labelled Abs is usually either alkaline phosphatase which converts 5 Bromo-4-chloro-indolylphosphate (BCIP) substrate into a blue product. or horseradish peroxidase with H 2 O 2 as substrate, oxidizes 4-chloro-1 naphthol into an insoluble blue product. an alternative approach to detect horseradish peroxidase is to use chemiluminescence. In the presence of H 2 O 2 and luminol, HRP oxidizes luminol with emission of light. the light emission can be detected by exposing the blot to a photographic film.

17. Markers for Western blotting Enzymes are commonly used markers, 125 I labelled secondary Ab. Flourescein isothiocyanate labelled secondary Ab. Gold labelled secondary Ab. Biotinylated secondary Ab.

18. Radioactive detection: X-rays Radioactively labelled DNA can be used to detect DNA-binding proteins on a blot. The blot is first incubated in a solution of radiolabeled DNA, then washed and autoradiograph of the blot is made. The presence of radioactive bands detected on the autoradiograph, identifies the presence of DNA binding proteins on the blot.

19. Western blotting can also identify a specific Ab in a mixture . Known Ag of well defined molecular weight are separated by SDS-PAGE and blotted onto the membrane. Separated bands of known Ag are then probed with sample suspected of containing Ab specific for Ag. Rxn . of a Ab with a band is detected by using either radiolabeled or enzyme linked secondary Ab specific for Abs in the test sample.

20. Applications of Western Blotting The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease’). Some forms of Lyme disease testing employ Western blotting. Western blot can also be used as a confirmatory test for Hepatitis B infection . In veterinary medicine, Western blot is sometimes used to confirm FIV+ status in cats.