Immunoprecipitation - Bands in negative control (View forum version)



protein2

Posted 08 August 2009 - 06:23 PM

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?

miBunny

Posted 09 August 2009 - 10:18 AM

What size bands are you seeing and how many bands?

laurequillo

Posted 25 September 2009 - 02:57 AM

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?)