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Lysis buffer for Co-IP - (Jun/29/2012 )


i am trying to do a Co-IP. I lyse the cells in Tris-HCl , with NaCl, EDTA, EGTA, NaF, glycerol, triton X-100 and protease inhibitors. I resuspend the beads containing the IP product in Laemmli buffer. I am having problems with the Western blot results, and it's not due to problems with the Westerns because I have ran usual samples alondside and they are fine. The protein in some lanes isn't present from the input, IP or supernatant and the westerns look really streaky and generally horrible. When i stain with ponceau there no/little protein and it's a solid streak. I have been going over the protocol a lot trying to find problems. Retrospectively, the only problem i can see is with the input and supernatant samples. These are both in the lysis buffer and then after the IP, before I run the Western I add 0.1% B-mercaptoethanol and bromophenol blue and then i boil the sample for 5 mins. This is what i usually do with the Laemmli samples but can I not boil the samples in lysis buffer even after I've finished the IP? Would this degrade the protein so it cannot be detected it.

Thanks in advance


the triton may be displacing (or preventing binding of) the sds from (to) the protein


mdfenko on Fri Jun 29 16:18:06 2012 said:

the triton may be displacing (or preventing binding of) the sds from (to) the protein

Along those same lines, from my experience coming directly from a lysis buffer to SDS-PAGE produces bands that are not well resolved. It might be a good idea to do a clean-up step prior to running the gels, maybe something like a centrifugal ultrafiltration. If you are concerned with the triton-x, my company makes a product that is intended to act as a substitute for Triton-X. Our non-ionic acid labile surfactantshows good lysis activity, with the primary advantage being that you can degrade the surfactant and easily remove it from the protein samples so that it won't cause interference in downstream analysis.