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confirm proteins interacting with histones using acid extraction


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#1 Catherinewang

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Posted 01 July 2011 - 12:12 AM

Dear all,

I am doing an experiment to confirm the interaction of my protein with histone H4. I tagged the protein with 3HA, and did the immunoprecipitation using the HA beads; however, I failed to see the interaction between my protein of interest with histone H4. Is there any suggestion that how I can perform this experiment properly? Since histones are highly basic, shall I use the acid extraction instead of the HA-purification? If I can detect the interaction by using acid extraction, is that sufficient to say that these two proteins have physical contact? Is there any positive control proteins that have proven to physically interact with histones using this way? I really hope that someone can help me out....Thanks a lot in advance! :rolleyes:

#2 chabraha

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Posted 01 July 2011 - 05:33 AM

I think a histone extraction would be way to intense of a treatment to detect an interaction..........Instead try enriched for a chromatin or nuclear fraction before performing the IP. In my experience using a non-ionic detergent in the lysis of isolated nuclei followed by a light sonication step works well. As for a positive control CBP and p300 both acetylate H4 and could possibly be detected in a interaction with H4...........Otherwise you could always isolate your protein of interest using an antibody, elute it from the antibody and perform a binding assay with recombinant H4 or nucleosomes.
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#3 knuf

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Posted 01 July 2011 - 05:34 AM

While histones are acid-stable, I would guess that your protein of interest likely is not and thus, wouldn't co-extract.

#4 Catherinewang

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Posted 05 July 2011 - 12:25 AM

Thanks a lot for your suggestions! So if I try to enrich chromatin fraction followed by performing the IP, will the buffer condition (for example, the pH and salt concentration)affect the binding? Would you mind providing me the details about the sonication steps? Performing a binding assay with recombinant H4 seems to be a good option, the thing is I am not sure how to do it and I am working with fission yeast.
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#5 chabraha

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Posted 05 July 2011 - 09:17 AM

pH and salt always affect the IP..........try lysing the cells in a neutrally pH'ed (7.4 or so)hypotonic buffer, gently spin down the nuclei and then just hit the nuclei with an appropriate amount of IP lysis buffer (pH 7.4-8.0) containing ~150mM NaCl, some EDTA, 1%Triton or 0.5% NP-40.....or both and just lightly sonicate to crack the nuclei open and shear the DNA.......maybe say, 10 X 1sec pulses at 20% power or less.
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