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thank you again for your help previously in designing the parameters to run a phospho-b4 integrin elisa. I tried to run this and had some problems, and I suspect it may be due to the lysis buffer I am using. Do you have any suggestions for what lysis buffer to use for this kind of a membrane protein? I am guessing that I want something that doesn't denature the protein, but releases it from the membrane fraction - would RIPA buffer work for this? Or do you have a suggestion of what other lysis buffer could work to release the phospho-b4 integrin and be compatible with an ELISA? Thanks!
Asked on Oct 22 2012
Thanks for your email.
NP-40 and RIPA buffers are good for solubilizing membrane proteins. Please see our western blotting guide for some further details and buffer recipes at https://www.abcam.com/index.html?pageconfig=resource&rid=11375
It might also be necessary to use the extraction buffer as a wash buffer. A typical wash buffer is just TBST or PBST. Washing with TBST or PBST may make the membrane protein "unhappy" such that it is not soluble anymore or does not retain expected function.
Also be sure to include phoshatase inhibitors in the extraction buffer.
I hope this is helpful. Please contact me again if you have any further questions.
Answered on Oct 22 2012