Western blotting is a powerful tool for studying proteins. A popular technique invented by everyone from freshmen to skilled researchers, western blotting has a number of purposes.
It is generally useful for identification, semi-quantification (can indicate relative levels of a protein, but not absolute size), and for determining the size of a particular protein. You can visit www.bosterbio.com/services/assay-services/western-blotting-service to get an affordable western blotting service with a fast turnaround time.
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Blotting is sometimes named immunoblotting or protein blotting because it uses antibodies to detect specific antigens. The "absorption" part refers to the process of protein transfer from the gel to the membrane.
The essence of Western blotting is to take a sample containing a protein mixture (natural or denatured) and separate the protein by gel electrophoresis.
Secondary antibody, which can be visualized (eg, by staining or immunofluorescence), is added and bound to the primary antibody. If everything goes according to plan, the result should be an image with a clear and distinct band representing the target protein.
1. How should I prepare the sample?
If using a tissue sample, it must first be divided by mixing or homogenizing it. Add buffer to allow cell lysis and solubilize protein.
Protease and phosphatase inhibitors can be announced into buffers to avoid protein breakdown.
2. What kind of gel should I use?
Maximum Western blots use polyacrylamide (PA) gel together with sodium dodecyl sulfate (SDS) buffer. Western blots that use denatured proteins typically use a kind of electrophoresis known as SDS-PAGE.
SDS causes a negative charge on all proteins, which allows them to be separated by molecular weight during electrophoresis. Gel width can affect the quality and quantity of protein detection.
3. Do I have to use a blocking agent?
After electrophoresis, the protein is transferred to the membrane so that it can be detected by antibodies. An inhibitory agent is then used to prevent non-specific binding between the primary antibody and the membrane.
Skimmed milk powder or cow's serum albumin together with a small amount of detergent is often used as inhibitor.