Immunoprecipitation - Bands in negative control - (Aug/08/2009 )
Hi, I am having trouble with an IP.
In my most recent experiment I pulled down my protein using a rabbit antibody, and included a negative control that contained only my antibody and beads in the IP buffer (no lysate). Then I used a mouse antibody to detect my protein by Western blot.
In my IP samples I detected bands at the expected molecular weight, but I also detected identical bands in the aforementioned negative control. I got the same results when I switched to different antibodies (IP with mouse antibody and WB with rabbit antibody).
My other negative control is clean (lysate + IgG + beads). Is it possible that both antibodies I used for IP contain some peptide in the antibody prep?
What size bands are you seeing and how many bands?
If you see a band in the sample without lysate most likely is gonna be the IgG. What is the size of the band (around 55?)