Western Blotting involves following steps:
- Protein Extraction /Sample preparation
Cell lysates are most common form of sample used for western blot. Protein extraction is done in a cold temperature with protease inhibitors to prevent denaturing of the proteins. As tissue sample display higher degree of structure, homogenization or sonication is needed to extract the proteins.
After extraction, concentration is determined using spectrophotometer. & it is diluted into a loading buffer, containing glycerol which helps sample to sink easily into the wells of the gel. A tracking dye (bromophenol blue) in buffer helps to see how far the separation has progressed. The sample is heated after it is diluted into a loading buffer, in order to denature the higher order structure, while retaining sulfide bridges. Denaturing high structure ensures that negative charge of amino acids is not neutralized, enabling the protein to move in an electric field.
Western blot uses two different types of agarose gel: stacking and separating gel.
Higher, stacking gel is slightly acidic (pH 6.8) and has a lower acrylamide concentration making a porous gel, which separates protein poorly but allows them to form thin, sharply defined bands.
Lower gel, separating gel, is basic (pH 8.8), and has a higher polyacrylamide content, making the gel’s pores narrower. Protein is thus separated by their size in this gel, as the smaller proteins travel more easily and rapidly than larger proteins.
Proteins when loaded on the gel have a negative charge, as they have been denatured by heating, and will travel toward the positive electrode when a voltage is applied. Samples and marker are loaded into wells, and the empty wells are loaded with sample buffer. The gel is then connected to power supply and allowed to run. Voltage is very important, as high voltage can overheat and distort the bands.
After separating protein mixture, it is transferred to a membrane. Transfer is done using an electric field oriented perpendicular to the surface of the gel, causing proteins to move out of the gel and onto the membrane. The membrane is placed between gel surface and positive electrode in a sandwich which includes fiber pad (sponge) at each end, and filter papers to protect gel and blotting membrane.
The most important step in blotting is:
- The placement of the membrane between the gel and the positive electrode
- The close contact of gel and membrane to ensure a clear image
Membrane must be placed such as negatively charged proteins can migrate from gel to it. This type of transfer is called electrophoretic transfer, and can be done in semi-dry or wet conditions. Wet conditions are usually more reliable as it is less likely to dry out the gel, and is preferred for larger proteins.
Blotting can be achieved by either of two processes:
- Capillary Blotting
- Electrophoretic blotting
There are two types of membrane: Nitrocellulose and PVDF. Nitrocellulose is used as it has high affinity for protein and has retention abilities. However, it is brittle, and does not allow membrane to be used for reprobing.
PVDF membranes provide better mechanical support and allow the blot to be reprobed and stored. However, the background is higher in the PVDF membranes and therefore, washing carefully is very important.
Probing and detection (Washing, blocking and antibody incubation)
Blocking is a very important step of western blotting, as it prevents antibodies from binding to the membrane nonspecifically. It is often done with 5% BSA or Nonfat dried milk diluted in TBST (Tris buffer saline with Polysorbate 90) to reduce background. However, milk proteins are not compatible with all detection labels.
BSA blocking solutions are preferred with biotin and AP antibody labels, and antiphosphoprotein antibodies. Since milk contains casein, which is itself a phosphoprotein and biotin, it may interfere with the assay results. It is better to incubate the primary antibody with BSA since it is usually needed in higher amounts than the secondary antibody. Putting it in BSA solution allows the antibody to be reused, if the blot does not give good result.
Washing is very important as it minimize background and removes unbound antibody. However, membrane should not be left to wash for a really long time, as it can also reduce signal.
Detection is made using label antibody, usually with an enzyme such as horseradish peroxidase (HRP), which is detected by the signal it produces corresponding to the position of target protein. This signal is captured on a film which is usually developed in a dark room.
Data produced with a western blot is typically considered to be semi-quantitative. This is because it provides a relative comparison of protein levels, but not an absolute measure of quantity.
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