my question is when i find the concentration of protein by using microplate reader, then how to calculate the quantity of protein for WB?
The amount of protein to run in a lane of SDS-PAGE for use in Western blotting depends on the sensitivity of the Western blot, which depends on multiple factors, including the proportion of the total protein sample composed of the antigen, the affinity of the primary antibody and the method of detection. You will have to do some experiments to find the right amount of protein to load.
I agree with Adam! It depends on how abundant the protein in in your sample(s). If it is very abundant, a small amount will suffice (10ug for example, but you could load less if very abundant). If the protein is less abundant, you will probably need to load more. You could do an optimisation experiment? Load different amounts (for example 20, 40, 60, 80ug) and see what you get?
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