problems with protG-agarose beads? - (Oct/19/2006 )
Hi,
I have a post about problems with the PCR from chIP material. Now I'm thinking about a possible explanation and I would like to know if somebody also had this same problem before.
The thing is that in my chIP experiment, when I perform the PCR using my specific pair of primers I get a unique and nice band when using the inputs, and the ch-immunoprecipitated material using an antibody against one transcription factor. The problem arise when I ch-immunoprecipitate against a different transcription factor, because this material give me two bands in the PCR, the specific band and another one that seems to the double size. Thinking in the possibilities, I realize that the problematic immunoprecipitation was done using protG-agarose beads, when the rest were done with protA-agarose beads. Is anybody facing the same problem or similar when using protG instead of prot A-agarose beads? I use the ones from Upstate.
Thank you
I have a post about problems with the PCR from chIP material. Now I'm thinking about a possible explanation and I would like to know if somebody also had this same problem before.
The thing is that in my chIP experiment, when I perform the PCR using my specific pair of primers I get a unique and nice band when using the inputs, and the ch-immunoprecipitated material using an antibody against one transcription factor. The problem arise when I ch-immunoprecipitate against a different transcription factor, because this material give me two bands in the PCR, the specific band and another one that seems to the double size. Thinking in the possibilities, I realize that the problematic immunoprecipitation was done using protG-agarose beads, when the rest were done with protA-agarose beads. Is anybody facing the same problem or similar when using protG instead of prot A-agarose beads? I use the ones from Upstate.
Thank you
Maybe you have some contaminating DNA in the protein G beads that your primers just happen to be able to amplify? Sounds pretty unlikely but it's easy to check. Just do a sample with beads only (no chromatin).