Protocol for histone acid extraction for Western blot - (Aug/22/2005 )

hello!

desperately need some good protocol for histone acid extraction (for western blot)!

thanks in advance

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Hey Annat
USe the protocol described in Eur. J. Biochem. 268, 5424-5429 (2001). Works pretty good

All the best

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Abcam have an excellent, simple protocol for acid extracting histones from mammalian cultured cells:

http://www.abcam.com/assets/pdf/protocols/...%20protocol.pdf

Hope this helps :)

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These protocols are all good but if you’re going to do anything but a western I’d recommend an H2SO4 extraction followed by dialysis. Check out UpState’s pan anti-AcH3 and AcH4 antibodies for a good protocol. Some authors mention a TCA precipitation following H2SO4 extraction, but I’ve never been able to dissolve those pellets in dH2O as the authors claim. Cheers.

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Hello everyone,
I have not done Western to detect histones, but I might need to do it in the near future.
I was wondering what the significance of the acid-extraction step is. What happens if you just run total proteins in the SDS-PAGE sample buffer in the ordinary way?

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acid extraction dissociates all the histones within a nucleosome. without it, on a western to detect histones, you will get abberant band sizes that do not equate to each individual histone, this is simply because the conventional extraction process does not dissociate the nucleosome complex into individual histones, acid extraction does.

Nick

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Hi Nick,
One more question.
In acid-extraction, how do proteins other than histones behave? Do they retain the same relative abundances and the appparent sizes?
Thanks.

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namekuji,

that is a very good question and I don't know what the answer is as biochemistry is my weakness.....must meditate on this....


Nick

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I’ve been doing acid extractions for a while now and it’s been my experience that there are relatively few proteins, except other chromatin associated and basic proteins, in the acid soluble fraction. I don’t do much with the acid insoluble pellet because the acid extraction itself is denaturing and without properly removing excess acid from the pellet, it’s hard to say what state the proteins are in. I’ve tried to resuspend the acid insoluble pellet in Laemmli buffer directly but to little avail.

statler

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sorry, I don't agree. You can easily get individual histones by RIPA extraction or direct lysis in SDS-PAGE smaple buffer. The main advantage of acidic extraction is that you enrich the histones. With respect to westerns you can therefore load much more histones per lane as compared to total extracts (which is required for many modified-histones-antibodies which are very insensitive)

after acidic extraction with HCl, I also recommend dialysis against acetate after which you can lyophilze your sample and reuse it for either western but also resuspend it and analyze them in non-denatured conditions.

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