Nuclear extract troubleshooting - (Mar/13/2011 )
My protocol to separate nuclear and cytoplasmic proteins is based on hypotonic cell lysis followed by Nonidet P-40 treatment and recovery of nuclei by centrifugation. However, after that I can't suspend nuclei pellet in glycerol-containing buffer, even after pippeting and vortexing. Using a T-75 flask (~4 million cells), this protocol yielded ~20ug of nuclear proteins. Is it normal? Why is nuclei pellet insoluble? Should I sonicate it? Help me to solve my problem. Thanks.
this is covered in another forum.
I found if you heat it a little it will break it up ok.
it is probably DNA stuck together...