07 January 2021 4 6K Report

I am currently working on a kinase protein which is only present within the nucleus and need to perform a Co-IP to confirm its binding with another protein. I have consider a lot of lysis method available and try it on my own but I guess those weak ones (typically with 1% Triton/NP40) doesn't work very well on me. I tried to apply weak sonication to the cell lysate ( 40W, 3-4 pulses) and the nucleus is not well lysed as shown on western. Another method I tried is RIPA buffer but there is a concern that it might be too strong to disrupt the protein interaction that I am going to test, and I have seen in some other discussion that RIPA is not a good choice particularly with kinase protein.

I am currently considering some other physical method such as gauge needles or freeze thaw but I ain't sure if they are good choice especially I need to break the nucleus open. I wonder if you guys have any suggestions on that (the main goal is to break nucleus open while maintain the protein interaction). Which detergent should I better choose (Triton X-100, NP40, SDS)? And any other physical method recommended?

In case Co-IP is not the best choice, any other assays should I perform (currently thinking of confocal microscopy for co-localization?) to achieve the same purpose?

Thanks.

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