NP40 concentration in protein lysis buffer - (Apr/07/2005 )
I noticed our protein lysis buffer (use for lysis of most tumor cell lines, Hela, 293, SAOS2, etc) is 50mM Tris (7.4), 250mM NaCl, 5mM EDTA, 0.1% NP40, plus protease inhibitors. And I compared with others. Most use 1% NP 40. But our yield is good, and we look at nuclear proteins, still good. And some people use SDS in the lysis buffer. Just wonder whether it matters a lot. Anybody has some idea or experience?
I am surprised your results are good! Generally people do use 1%NP-40 and still have trouble isolating nuclear proteins. The cell lines you are using HeLa and 293 (not sure about the other one) are VERY highly transformed and as such have a VERY high nuclear to cytoplasmic ratio. Just about any lysis buffer would give a good nuclear yield with these cell lines. Why not try the 1% and compare it to 0.1%? If your buffer is working though and you are happy with the results, why change?