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Chromatin isolation


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#1 rkay447

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Posted 23 March 2009 - 09:15 AM

I'm hoping someone can help me with an assay I've been trying to work with for awhile now. I'm trying to look at the chromatin binding of DNA replication proteins under specific conditions. I am using the highly published protocol from Mendez and Stillman whereby chromatin is isolated by use of buffers and centrifugation. I'm having great results with the experiment and my controls of HU and Nocodozole blocked populations but can't seem to get the DNase control to work. My chromatin sample is not sticky and easily pipettes after DNase treatment but the proteins still come up as being in the chromatin fraction (even after extensive washing of the chromatin sample after digestion). I'm so lost and confused. I know the digestion is working since the sample is no longer gooey and can be pipetted but why aren't the proteins coming off the DNA?? I also know that I really am isolating clean chromatin since my HU and Noc samples show the proper chromatin association of my proteins of interest! Really need help!! Thanks for any suggestions!

#2 rkay447

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Posted 26 March 2009 - 08:12 AM

bummer

#3 mikew

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Posted 26 March 2009 - 03:30 PM

Hi,

Do you do a control to probe for your proteins in the soluble vs. the chromatin fraction? Do your proteins show up only in the chromatin fraction? Maybe they are being lost during the extraction process.
Are you sure your protein should "come off chromatin" after DNAse? Maybe it stays on?
Did you do a DNA gel with your DNA after digestion? The times I have done
DNAse digestion I have always run a sample on a gel to ensure it was cut to mono-tri nucleosome length.
My experience with DNAse digestion is this:
I have been IPing chromatin bound proteins. If I compare high salt extraction vs. DNAse treatment (using the Stillman isolation method) I see no difference. In conclusion, high salt extraction releases chromatin bound material in my hands, pretty much as well as DNAse treatment.
Furthermore, the chromatin-bound fraction is quite small. You need a boat-load of material to detect chromatin-only proteins (using the Dnase-EDTA-EGTA extraction method).
Anyways, hope this info can help somehow.




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