Hello, I want to ask about the method of analyzing phospho form and total form of proteins by western blot.
I learned that many people conduct stripping the membranes to study expression level of phospho form and total form. But personally i prefer analyzing both forms at the same time so i cut the membranes into pieces(I cut the membranes at nearly the center parts of the size marker proteins to avoid cutting the sites where proteins originated from the cells exist) so i can see both forms at the same time because these pieces can be incubated with different antibodies in respective trays.
But it seems that most people choose stripping but i want to know whether even my method can be another good method. (To sum up, i think i can see p-form proteins and total form proteins at the same time by incubating different parts of the membranes.)
And additionally, i want to ask whether my method can also be applied to proteins even though they are not related to analysis of p-form and total form of proteins.
I would prefer to determine the expression levels of proteins the same way in which you have described. Stripping, however gentle it might be, can lead to loss of some protein from the membrane. If you are trying to quantify the levels of phospho proteins versus total protein, cutting the membrane and blotting against the respective antibodies seem very reasonable to me.
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