Hey everyone,
I've had some problems with my protocol of nuclear fractionation and this forum was recommended. The protocol is based on hypotonic cell lysis with RBS followed by Nonidet P-40 treatment and recovery of nuclei by centrifugation. However, after that I can't suspend nuclei pellet in appropriated 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 or use a motor pestle? Help me to solve my problem.
Thanks.
Yes, Aaron, but due to excessive bureaucracy to import scientific goods in Brazil, sometimes it's easier troubleshooting, unfortunately. For example, we have to plan our antibodies or drugs needs with, at least, six months in advance. Anyway, thank you for your suggestion, I gonna look for these kits.
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