Hi all,
I am performing a Co-IP experiment where would like to check the binding of my protein to the DNA of interest. I kept a control where I incubated the DNA and antibody only with the protein A/G beads, after which I performed PCR to amplify the DNA of my interest.
I am seeing the desired PCR bands even in the control. Is it possible that the protein A/G beads show some sort of nonspecific binding to my DNA of interest resulting in these bands? I found the following links mentioning nonspecific binding of DNA to Co-IP beads:
Article Chromatin Immunoprecipitation to Analyze DNA Binding Sites of HMGA2
https://wiki.zfin.org/display/prot/Chromatin+Immunoprecipitation+%28ChIP%29+Protocol+
https://cdn2.hubspot.net/hubfs/266350/Whitepapers/How_to_plan_an_immunoprecipitation_of_Myc-tagged_proteins.pdf?t=1496396328227
Any help is appreciated!
Thanks!
Hello Dr Subramaniyam,
I think before putting up the reaction you should first add beads to the protein and allow it on rotation for 20 minutes. Discard these beads and transfer the supernatant in a new eppendorf and now begin your experiment with your desired antibodies.
I believe this will work out for you.
Good Luck and Regards,
Pratibha
Hi Prathiba,
I might have missed something in your reply. The purpose of my experiment was to ensure that the DNA pull down is not just due to the agarose beads nonspecifically pulling down the DNA and to show that it is mediated by my protein. So I fail to understand how adding beads to the protein and discarding them can give me the picture.
Can you please elaborate on your answer?
Thanks,
Subramaniyam
Hi Subramaniam,
Performing with this will remove out the non specific binding of the protein with the beads. In any case, I perform this step in my Immunoprecipitation experiments.
I guess, the bands you are getting may be the reason of non-specific binding of the proteins with the beads. I hope you understand this point.
Thanks!
Pratibha
Hi Pratibha,
I have not added my protein to the mixture at all. The mixture only contains the beads, antibody, and DNA. The purpose is only to check whether the DNA can nonspecifically bind to the agarose beads.
Thanks!
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