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Bands are lower than actual protein - (Aug/31/2010 )


I'm doing a Western to quantify the activity of P21-activated kinase (PAK) by using a phospho-serine/threonine antibody. I first pulled-down alpha-PAK with an antibody and then added Protein G Agarose beads. I then run the samples and total cell lysates on a 10% gel and immunoblot with the pSer/Thr antibody for the samples and alpha-PAK antibody for total cell lysates (as a loading control). My problem is that my pSer/Thr bands do not match up with the total PAK bands. PAK is 65 kDa and is on the right side of the blot I have attached. Does anyone have any suggestions or ideas of what the problem may be?

Attached Image


Multiple epitopes to one antibody and not the other? Quantitative westerns are a tricky proposition...