Hi,
Which lysis buffer would you recommend to make a protein extract from tissue that does not interfere with immunoprecipitation afterwards? I'm currently using RIPA + protease inhibitors, however I'm not sure it negatively affects my IP.
Kind regards,
Stijn
Hi, I suggest you to use RIPA (you may want to avoid SDS expecially if you want to keep protein-protein interactions, but you can add deoxycholate instead) for tissue lysis and dilute it before performing the immunoprecipitation. You can dilute it with a buffer containing only Tris and NaCl to keep their concentration but reduce (maybe 4 times) the detergent concentration. Good luck
I agree and would also propose to use a Tris-buffered solution as mentioned before.
For immunoprecipitation, it is common to use a lysis buffer containing 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, and a protease inhibitor such as aprotinin, leupeptin, or pepstatin.