I have purchasehuman histone octamers from Sigma (SRP0408) for nucleosome reconstitution experiments. I would like to check the protein complex in octameric form. Running it on SDS-PAGE will nature the protein and hence it will not exist in octameric form. And I heard that native PAGE will not work for this purpose due to the charges on histones.
Please let me know how do I detect the octameric form.
Superdex 200 chromatography in 2M NaCl is the typical standard. Octamers are not stable at physiological ionic strength...
Histone octamers exists only at high salt (2M NaCl); at lower salts dissociate to dimers (H2A-H2B) and tetramers (H3-H4). 1. Check the stoichiometry and purity by loading 3-4 ug of total histones on18% SDS PAGE. Next, adjust your histone mix to 2M NaCl (KCl) by dialysis. 2. Reconstitute nucleosomes at a minimum 5 ug scale by gradient salt dialysis with a piece of 150-250 bp of the Widom positioning 601 DNA. 3. Check the reconstitution quality on a small 5% native PAGE-0.25x TBE buffer - it should migrate as a single band. Note that Superdex 200 chromatography is a very gourmand tool. One advice - if you want to go seriously into that business you should be able to prepare yourself recombinant histones, not using commercial ones.
If you want to reconstitute core nucleosomes first you must use a defined DNA + histones etc and then check stochiometries of the bound octamers by various approaches. There many protocols that you can find. .
Any histone equimolecular histone solution will spontaneously fold into octamers at high salt and neutral pH, so you can use it for nucleosome reconstitution experiments.
Hi Dr. Dimitar,
What are the gel running conditions for histone octamers to be checked on 18% SDS-PAGE?
Could you provide me a detailed protocol or a reference for the same?
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