I am studying a post translational modification through Co-IP and I would like to perform a denatured IP to confirm that my results are not from post translational modification of a different associated protein. I have spent a lot of time finding the optimal buffer for my IP antibody (50mM Tris, 150mM NaCl, 1%NP-40). I have ready that most denaturing protocols utilize high concentrations in a small volume of something like SDS or BME followed by heating and then the diluting the sample before using it in the IP.
I'm concerned my IP will not work every well in a different buffer. Would it be enough to just heat the sample? Or could I treat the NP-40 in my buffer similar to the SDS (use a higher concentration + heat and then dilute it)? Any suggestions would be helpful
Thanks!
Anna
Hello Anna. If you just heat your sample, you will likely see a lot of protein aggregating and precipitating. That will defeat the purpose of your experiment. Could you try denaturing at high protein concentration in your IP buffer supplemented with, for example SDS and 2-ME, heating, and then diluting using your IP buffer without the dentaturing agents? You need to reduce their concentration in order to prevent damage to your IP antibody or interfering with antibody-antigen interactions. Another caveat would be that your primary antibody may not recognize its target epitope in its denatured state.
regards, Bruce
Hi Anna
To give you an answer I need to know more about the experiment.
What is the modification you are looking for?
Is the protein overexpressed or endogenous?
Is a membrane, cytoplasmic or nuclear protein?
Do you have a specific antibody for detection (e.g. phospho specific antibody), do you use a pan antibody or do you check by shift in SDS-PAGE?
As Prof. Allen already mentioned, using any denaturing agents or heating the sample before the IP is not a good idea. You will face a lot of aggregation/precipitation and your antibody might not work well.
With these information I can help you to design the buffer you need.
Best,
Edoardo.
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