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The Western Blot technique is a research method specific to the laboratory that is used to identify some specific groups of proteins in a mix of proteins. The method is also used to examine the size of selected protein molecules and also to quantify the number of the desired protein molecules. This method was named after the previously discovered method called the “southern blot” technique, as both of the methods are very similar in nature.
The Western blot technique
The Western blot technique consists of several steps, which are,
The primary step of the western blot technique is to make a mixture of proteins by adding a sodium dodecyl sulphate (a detergent). It coats the protein with a charge that is negative and helps the protein unfold into linear chains. Next, a procedure referred to as gel electrophoresis is applied to distinguish between the protein molecules with respect to their size. The protein molecules are shifted to the blotting membrane from the gel. The term “western blotting” is derived from this step. After the transfer procedure has finished, the membrane takes all of the bands of protein that were at first on the gel. After that, a procedure called “blocking” is applied to the gel. It prevents any irrelevant reactions from happening.
Next, the membrane is brooded with an antibody referred to as the “primary antibody.” It specifically binds the selected protein. After that, the primary unbound antibody is cleaned away from the membrane. After this, the membrane is brooded again with the secondary antibody that recognises and significantly binds the second antibody. After that, any unbound antibody is washed away. The second antibody is connected to a reporter enzyme. This reporter enzyme often produces light or colour, so it is pretty easy to identify. It can also be easily photographed and images for this reason. So, in this way, a specific group of proteins can be distinguished from a mix of proteins.
The principle of the Western blot technique
This technique points to the protein molecules, and these molecules are shifted to a membrane that is hydrophobic in nature (a membrane that is water repellent). This membrane distinguishes the protein molecules by using some specific antibodies. As mentioned earlier, the membrane is kept on the gel and separated protein molecules are separated and transferred electrophoretically on this membrane.
The enzyme is mainly used to find out the target protein and is imaged by the chromogenic method. In this method, the separated protein molecules are transferred to a membrane from the polyacrylamide gel using a machine called the blotting apparatus. A partially dry or a system of tanks is used for shifts. In a semi-dry system, the blotting period is short, but in a tank system, the blotting period is long.
The buffer volume in a semi-dry system is small, but the buffer volume in a tank system is large. In a semi-dry system, the transfer efficiency is poor for large-sized and high-weight protein molecules. In a tank system, the transfer efficiency is good for high-weight protein molecules, and these molecules are evenly transferred. For membranes, PVDF and nitrocellulose membranes are used. The membrane of PVDF is expensive, but it has a higher absorption capacity and is much stronger.
These two attributes make it eligible for the straining process. This substance can detect alkaline phosphatase. Nitrocellulose membrane, though fragile, is also cheaper. This membrane is useful for re-probing (re-probing refers to the stripping of the antibody membrane after probing and detecting it with a second antibody). Various buffers are used for the transfer process. The nature of the buffer depends on the weight of the molecules of the protein molecules and the nature of the selected protein. The make-up of the most commonly used buffer is as follows:
25 mM Tris-HCl
Glycine (192 mM)
MeOH (at 20%)
The methanol helps to stabilise the given gel and prevent it from swelling. Reducing the concentration of methanol helps the gel to swell a little, which induces the protein transfer while transferring high-weight protein molecules. The probing and blocking are done by using antibodies. The word “blocking” means preventing the binding of non-specific antibodies by the membrane. Various kinds of blocking elements are used, such as 1-3 percent BSA in PBS-T (BSA means Bovine serum albumin), 1-5 percent skim milk in PBS (as skim protein has the phosphoprotein casein, so it can’t dictate phosphoproteins), etc. The word “PBS” refers to phosphate buffered saline, and it contains 0.05 percent Tween 20.
After blocking, the membrane is examined with both primary and secondary antibodies. But the concentration amount of antibody that is primary is critical as if it is too low, it can’t detect the selected protein, and if the concentration gets too dense, many reactions occur and the detection of the wrong protein molecules is assured. When one is conducting the experiment the first time, one must determine the concentration according to the datasheet. If the desired result is not obtained, several thinner versions of the primary antibody are used. A different thinning buffer is used if that doesn’t work.
Conclusion
The western blot technique is an important biological process that is used for protein detection. This method is extremely important and widely used in immunology. This is a laboratory-based method and requires precision. For all these reasons western blot technique is important for different public service exam aspirants.
