Why my target protein did not show up, but the non-specific binding showed up - (Jun/27/2008 )

I did western blot of the same sample twice. The first time, the target protein had very very strong signal, but there were some less strong non specific binding. The second time, I reduced the protein at half concentration. There were almost no signal for the target protein (60KD), but the non specific protein above the target showed up (not very strong either). However, the control beta actin was very strong as before.

Any thoughts will be appreciated.

Susan

the reasons which i can think of are 1. are the lysates properly stored(without several freeze thaw cycles) 2. antibody is contaminated or degraded accidentally. 3. not enough target protein in your lysate.