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.
Amended on 6 November 2017
I have not yet purchased Histone (Sigma), but our HPLC-Surf-SEC method has indicated that Histone H3.1 (detected in hepatoblastoma and retinoblastoma) elutes as homo-tetramer, and Hemoglobin A (HbA; hemoglobin α2β2) elutes as hetero-tetramer. Then, we have found that Histone H3.1 only (other type of Histone is not present) is present in the fetal hepatocyte Hc cells, and is the protein of highest amount (please see file; HepG2 fucoidan). Adult Histone (histones H2A, H2B, H3, and H4) is detected in adult hepatoma HepG2 cells. Interestingly, fucoidan has not changed the Histone-gene expression. Histone H3.1 seems to be linked to development, but Adult Histone (histones H2A, H2B, H3, and H4) seems to be linked to differenciation. Adult Histone may elute as hetero-octamer. Please, try this quick and reroducible HPLC-Surf-SEC method (analysis time is only 70 min).
By the way, Sigma Histone (SRP0408; histones H2A, H2B, H3, and H4) has N-terminal His-Tag, and may be eluted other than hetero-octamer.
Further, it is noteworthy that only one liver tissue with liver cirrhosis (named as No.6) has expressed Histone H1.0 (H1') at 0.39 μg/mg tissue protein and Histone H1.1 (H1a) at 1.31 μg/mg tissue protein. This unique gene expression may be related that this tissue uniquely has L protein of Marburg virus (MARV).
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?
Usual 18% SDS PAGE as described in Maniatis. Run at 18 mA fixed current. Stop the gel 15'-20' after the loading blue (BB) is out of the gel. You can use also commercial 15% SDS PAGEs. You can find many detailed protocols on
Google, see f.e.:
Article Rapid Purification of Recombinant Histones
https://www.google.fr/url?sa=t&rct=j&q=&esrc=s&source=web&cd=17&cad=rja&uact=8&ved=0ahUKEwjoiPOWvbbXAhXRo6QKHS1zAjg4ChAWCEcwBg&url=http%3A%2F%2Fweb.stanford.edu%2Fgroup%2Fgozani%2Fcgi-bin%2Fgozanilab%2Fwp-content%2Fuploads%2F2014%2F01%2FExpression-and-Purification-of-Recombinant-Histones-and-Nucl-from-Chromatin-Protocols.pdf&usg=AOvVaw3tVCMMqNCpR-jE6hNEBKM2
http://web.stanford.edu/group/gozani/cgi-bin/gozanilab/wp-content/uploads/2014/01/Reconstitution-of-Nucleosome-Core-Particles-from-Recombinant-Histones-and-DNA.pdf
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