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Chromatin Protein IP


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#1 CoolMittens

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Posted 18 July 2011 - 08:51 AM

Hello

I was wondering what would be a way to IP protein that is specifically bound to chromatin. I do not need the chromatin itself, so no need for a ChIP assay (or crosslinking) but just the protein. We are attempting to see if our protein-chromatin interaction is stabilized with treatment.

Previously I had preformed nuclear extraction (hypotonic swell, cyto harvest, wash, wash, high salt nuc lysis, wash, wash, ->> DNAseI treatment to fragment the DNA/increase solubility -> IP all three fractions independently) but I do not know how pure/appropriate this is.

Additionally, any ideas for a loading control to test chromatin fraction purity?

Thanks in advance!

#2 chabraha

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Posted 22 July 2011 - 10:50 AM

Purity shouldnt be paramount...........you just want enriched fractions for the IP. When you do these enrichment/fractionation protocols it is very important to understand the chemistry involved. For example you say, "high salt lysis" what is high salt? compared to a hypotonic buffer 150mM salt is high.....but not high enough to extract the majority of core histones or heterochromatic foci. 250mM will extract chromatin bound proteins but still will not extract the majority of nucleososme bound core histones. To test for chromatin bound (I assume your refering to the high salt lysis step) purity.....maybe try H1 since it is salt extractable at low salt relative to core histones assembled into nucleosomes.........or try HDAC1........for your DNAse1 digested material......try H3 or HP1. Or use MNase instead and run a DNA gel to show that there is DNA present only in the final extraction.
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#3 CoolMittens

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Posted 28 July 2011 - 11:29 AM

Thanks!




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