Immunostaining non-hisstones protein in histone extracts - (Oct/30/2015 )

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?

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?

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)?

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