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Different intensity present in reference protein even though BCA performed - (Nov/21/2008 )

Dear all,

Recently I tried to do WB with a few protein extract samples from cell cultures havested by scraping. However, even with BCA assay performed and the amount of protein loaded is the same, the final reference protein, monoclonal anti-B-actin, intensity is not the same. Excluding that the BCA assay gives error or the WB performed is unpromising (i've repeated the procedure for a dozen of time already, poor me...), is there any possible reason that can explain this situation?



you may have lost protein during the transfer, some may not have exited the gel (yet) and some may have blown through the membrane.

also, depending on the actual readings, the bca may be off.