ChIP Fixation & Protease inhibitors - (Mar/11/2008 )
So after I fix my brain tissue in 1% Parformaldahyde/30 minutes at 4 degrees for ChIP, glyicine quench, I then homogenize the tissue in RIPA buffer with dounce homogenizer. I put PIC in the RIPA to block protein degradation.
My question - Is it really necessary to use PIC? I mean isn't everything kind of fixed together? I guess it's not totally fixed, so maybe it is necessary. What do people think?
-MKR-
QUOTE (MKR @ Mar 11 2008, 11:11 AM)
So after I fix my brain tissue in 1% Parformaldahyde/30 minutes at 4 degrees for ChIP, glyicine quench, I then homogenize the tissue in RIPA buffer with dounce homogenizer. I put PIC in the RIPA to block protein degradation.
My question - Is it really necessary to use PIC? I mean isn't everything kind of fixed together? I guess it's not totally fixed, so maybe it is necessary. What do people think?
My question - Is it really necessary to use PIC? I mean isn't everything kind of fixed together? I guess it's not totally fixed, so maybe it is necessary. What do people think?
I've found that the protease inhibitors make a big difference. I'm guess this is because the fixing is not complete so that some proteins are still active.
-KPDE-