immunoprecipitation troubleshooting - (Mar/07/2010 )
I performed an IP a few days ago, however my antibody failed to detect the protein of interest (148 kDa)in the IP samples and also the lysate samples. Instead i saw a large band at 90 kDa but i have put that down to non-specific binding. I blocked the blot using 5%milk (0.05% TBST) when incubating with the anibodies (overnight at 4 degress for my primary antibody and 1 hour incubation at room temp for my secondary antibody covered with aluminium foil). after incubation I would wash the blot for 5 mins with 1X TBST twice and the final wash with 1XTBS again for 5 mins. Could the length of washing be the problem? For me this seems like a an adequate amount of time for washes.
does anyone have some suggestions?
Your washes are not much for standard westerns. I do four 5 minute washes for each antibody step. Many protocols use 3 washes.
Could it be that the secondary is picking up the heavy chain of the antibody you used to IP?