3 replies to this topic

[eaton]

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!

[bob1]

both nuclear and cytoplasmic I would say, based on the lysis buffer recipe.  You still won't get the insoluble proteins though

[eaton]

bob1, on 15 September 2010 - 06:28 PM, 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 15 September 2010 - 06:28 PM, 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!