What portion of protein can I get under this protocol? - (Sep/15/2010 )
Hi! everybody:
I use tissue lysis buffer as 10mM Tris-HCl pH 8, NaCl 150mM, EDTA 1mM, nonidet P40 1%, SDS 0.1%, and 1X protease inhibitor cocktail. I mix lysis buffer and tissue together and crush tissue with pestle. Then, incubate the mixture on ice for 10 mins, freeze mixture at -70 degrees C, and thaw the mixture on ice again. After spin tissue debris down at 13000rpm, 5 mins. What portion of protein can I get? Only cytosol protein or both cytosol protein and nuclear protein? Thank you!
-eaton-
both nuclear and cytoplasmic I would say, based on the lysis buffer recipe. You still won't get the insoluble proteins though
-bob1-
bob1 on Thu Sep 16 02:28:00 2010 said:
both nuclear and cytoplasmic I would say, based on the lysis buffer recipe. You still won't get the insoluble proteins though
Thank you!!
And how can I adjust my lysis buffer to get these insoluble protein? Or I need another buffer to get the insoluble protein after this procedure? Thank you!
-eaton-
bob1 on Thu Sep 16 02:28:00 2010 said:
both nuclear and cytoplasmic I would say, based on the lysis buffer recipe. You still won't get the insoluble proteins though
Furthermore, if I want to separate the nuclear and cytoplasmic protein, what procedure can I do?
Should I use a different lysis buffer and different protocol?
Or can I do something after I getting these nuclear and cytoplasmic protein?
Thank you!
-eaton-