Western blotting

Western blotting

1. WESTERN BLOTTING PRESENTED BY: RAVI KUMAR M PHARMACY(1 st SEM) DEPARTMENT OF PHARMACOLOGY G H G KHALSA COLLEGE OF PHARMACY

2. BLOTTING Blots are techniques for transferring DNA , RNA and protein onto a carrier so they can be separated and often follows the use of a gel electrophoresis The These powerful techniques allow us to identify and characterize specific molecules in a complex mixture of related molecules. Some of the more common techniques include: Southern blotting (DNA) Northern blotting (RNA) and Western blotting (for protein)

3. WESTERN BLOTTING Western blotting, also known as immunoblotting or protein blotting, is a technique used to detect the presence of a specific protein in a complex protein mixture. It is a core technique in cell biology , molecular biology , virology and others. Western blots have become one of the most common analytical tools for the Detection of viral proteins. Characterization of monoclonal and Polyclonal antibody preparations. To determining the specificity of the immune response to viral antigens

4. WESTERN BLOTTING PROCEDURE FLOW CHART

5. SAMPLE PREPERATION All sources of protei n, from single cells to whole tissues, biological fluids and proteins secreted in vitro, are open to analysis by Western blotting In most cases, the cells are harvested, washed, and lysed to release the target protein For best results, all these steps should be carried out on ice This will minimize proteolysis, dephosphorylation , and denaturation , since all begin to occur once the cells are disrupted

6. The choice of extraction method depends primarily on the sample and whether the analysis is targeting all the proteins in a cell or only a component from a particular subcellular fraction The endogenous proteases may be liberated upon cell disruption and may degrade the target molecule the sample should be protected during cell disruption and subsequent purification by the use of protease inhibitors to avoid uncontrolled protein losses Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solublize proteins Tissue preparation is often done at cold temperature to avoid protein denaturing

7. GEL ELECTROPHORESIS The proteins of the sample are seperated using gel electrophoresis. Seperation of proteins may be by isoelectric point molecular weight, electric charge, or a combination of these factors The principle involved is the difference in the ELECTROPHORETIC MOBILITIES of different proteins Mix ingredients in the order shown above, ensuring no air bubbles form. Pour the separating gel into glass plate assembly. Overlay gel with water to ensure a flat surface and to exclude air. Leave to polymerize for ~ 20 minutes. Then prepare the stacking gel and pour on the running gel, insert comb and leave for 20 min.

8. SAMPLE BUFFER A sample of protein, is boiled in sample buffer (at 95 o C for 5 minutes) which contains: The β - mercaptoethanol reduces disulfide bonds SDS (Sodium dodecyl sulfate) disrupts protein secondary and tertiary structure Glycerol to make samples sink into wells Bromophenol Blue dye to visualize samples

9. The end result has two important features: All proteins contain only primary structure and All proteins have a large negative charge which means they will all migrate towards the positive pole when placed in an electric field. They migrate through a gel towards the positive pole at a rate proportional to their linear size

10. Loading Samples & Running the gel Samples are loaded into separate wells A protein marker is also loaded Run for 3 0-40 minutes Running Buffer ( pH 8.3) c ontains Tris Base Glycine SDS        

11. PROTEIN TRANSFER In order to make the proteins accesible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidine difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electro blotting and uses an electric current to pull proteins from the gel into PVDF or nitrocellulose membrane

12. BLOCKING Blocking is a very important step in the immunodetection phase of Western blotting because it prevents non-specific binding of antibody to the blotting membrane The most commonly used blocking solutions contain bovine serum albumin 3-5% (BSA) or non-fat dried milk in a solution of PBS (phosphate buffered saline) or TBS ( tris buffered saline) Often, a small amount of Tween 20 detergent is added to blocking and washing solutions to reduce background staining, and the buffer is known as PBST or TBST

13. ANTIBODY PROBING Once the protein samples are separated and transferred onto a membrane, the protein of interest is detected and localized using a specific antibody . The blot will be incubated in a dilute solution of antibody, usually for a few hours at room temperature or overnight at 4°C. The antibody is diluted in wash buffer (PBST or TBST) or in the blocking solution, the choice depends upon the antibody Since antibody preparations vary in their levels of purity and specific binding properties, there will be differences in the level of dilution required

14. Usually non-labeled primary antibody directed against the target protein. Wash the membrane several times in TBST while agitating, 5 minutes or more per wash, to remove residual primary antibody A species-specific, labeled secondary antibody directed against the constant region of the primary antibody is then used The secondary antibody serves not only as a carrier of the label but is also a mechanism to amplify the emitted signals, as many secondary antibodies can theoretically bind simultaneously to the primary antibody Secondary Ab is also diluted according to the manufacturer recommendations and incubated for 1 hour at RT

15. DETECTION WITH SUBSTRATE The most common antibody label used in Western blots is horseradish peroxidase ( HRP), a small, stable enzyme with high specificity and rapid turnover The signal is detected when HRP is exposed to a substrate solution in the final step of the immunodetection procedure Substrate solutions for Western blotting are chemical reagents that are acted upon by the enzyme to yield a signal that can be easily measured HRP label is typically detected with either colorimetric.

16. APPLICATIONS