I am looking to measure phospho-Erk and total-erk protein expression with western blotting, and am wondering if it is necessary to use a loading control. I normally measure phospho first, then strip and measure total on the same blot. Do I still need to normalize each to a loading control, or will that be redundant if I'm measuring from the same blot?
Thanks!
That depends on your research question. If "x % of my protein is phosphorylated, 100-x% not, and treatment changes this to y %" suffices, a loading control is not required. If, however, under treatment the expression of your protein (total) changes with respect to total cellular protein, then you'd probably want to catch that, too.
You could also use gels that retard phosphoproteins (doi:10.1002/elps.200800386), then you could detect the phosphorylated and unphosphorylated protein at the same time.
Also, recall that WB is, at most, a semi-quantitative technique.
Best practice would be to perform multiplex WB using fluorescent secondary antibodies. We at LI-COR routinely detect Pan-ERK and P-ERK using 680 RD and 800CW secondary antibodies. Under this design, you would be able to simultaneous incubation and detection and analysis without loosing any protein, due to stripping.
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