Coimmunoprecipitation using Ni-NTA, My non-his-tagged target bind to beads
Posted 13 January 2011 - 09:29 AM
I am frustrated by the CoIP recently. I am doing mammalian CoIP which I transfect protein A (with cmyc tag) and protein B (His tag)to 293 cells. I am using qiagen Ni-NTA agarose column.
Before I have done the Y-2-H screening, confirmed in the same system and then in vitro coimmunoprecipitation using TNT recticulocyte. Now come to the mammalian CoIP.
Please remember I have no problem in doing same CoIP several months ago. Beofore, the protocol is more or less the same as the Qiagen provided except that I have incubated the beads with 1% BSA for 1 hr before I added to lysate. However, since I have seen in the forum that the BSA in fact has no use in blocking the non-specific binding in the Ni-NTA agarose beads, so I stop using it.
I don't know if it is the reason behind but recently, I found that my protein A (has cmyc tag at N-term also His-tag at C-term BUT I have already added the stop codon in front of it and sequence has been checked) can bind to beads and being eluted out!!!! More weird thing is that I set up the transfection for the CoIP is like (1). Protein A plasmid + Protein B plasmid (2). Protein A + empty vector for roundup the plasmid amount. What I find out is the protein A band appear in the setup (2) is obviously stronger than (1). Moreover, the non-specific bands appeared were more than before. Theoretically, only protein B can be pulldown by Ni-NTA but not the protein A.
Anyone has any suggestions to me? I am really discouraged and don't want to do anymore, you know each time the experiment takes such a long time........
Posted 22 January 2011 - 01:22 AM
Posted 23 January 2011 - 01:14 PM
I remember using ProA-Agarose for some IP experiments and it seemed that the agarose could pulldown my protein as well was a few other proteins from the lab which we tested, nonspecifically.There was a report somewhere that agarose based matrices can do this to a lot of proteins (let me see if can dig out the reference).So, we used 5% BSA for blocking and it worked.Maybe your 1% is not enough.... alternately, using gelatin for blocking also works.
Thanks for the advice. But do you think that the concentration of 5% BSA is too high that excess BSA will still bind to the beads and is difficult to be washed off? Thank you