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.
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