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Immunostaining non-hisstones protein in histone extracts

histone extract western blot protein extract

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#1 Nadezhda Fursova

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Posted 30 October 2015 - 01:09 PM

Hi!

 

I have been extracting histones using 0.8M HCl. I have used acid-soluble fraction for SDS-PAGE and subsequent WB. I found that I can actually stain extract for other nuclear proteins - for example histone-modifying enzymes (H3K4 methyltransferases, RING1B). I wonder why I can see these proteins in the extract. I doubt that they are as positively charged as histones so they are some kind of background. I am not sure how specific it is. What fraction of these proteins is extracted with this method? Could it be considered a chromatin-bound fraction specifically? Is it similar to nuclear extracts? Any thoughts from people who understand better how these extractions work?



#2 mdfenko

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Posted 02 November 2015 - 05:41 AM

as long as the pH is not too close to their pI many proteins will be soluble in aqueous media.

 

have you tried staining the gel with coomassie or silver?


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#3 Nadezhda Fursova

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Posted 03 November 2015 - 05:48 AM

Hi,

 

Yes, of course, I stainded it with Coomassie. I can see a lot of protein bands on it. I just wonder if the protocol suggests that some fraction of proteins will be extracted more efficiently (like nuclear proteins or chromatin-bound proteins)?



#4 mdfenko

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Posted 04 November 2015 - 04:36 AM

when you crack open the nucleus you will also release other proteins which are soluble in the medium.


talent does what it can
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