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Nuclear extract troubleshooting

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#1 jonas albarnaz

jonas albarnaz


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Posted 13 March 2011 - 08:28 PM

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.

#2 sBrow



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Posted 10 May 2011 - 04:24 AM

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...

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