All the methods for in vitro nucleosome assembly (using gradient salt dialysis), I came across, uses purified H2A/H2B dimer and H3/H4 tetramer. Can individual histones (H2A, H2B, H3, and H4) in equimolar ratio be used for in vitro nucleosome assembly?
If so, please share the method (link or paper).
If not, please explain why it is important to use purified dimers or tetramers instead of individual histones.
I did some literature search and also contacted NEB tech support in this regard.
The nucleosome assembly requires correct protein complex formation that is usually in the cell achieved by the use of chaperones. In an in vitro nucleosome assembly assay using gradient dialysis method, the assembly of octamer will not take place by simply mixing the individual histones in the correct ratio. The histones have to be in the complex forms (H2A/H2B dimer, and H3/H4 tetramer) for in vitro assembly reaction. The H2A/H2B dimer and H3/H4 tetramer complex can be prepared using denaturation and refolding of histones.
For actual method, see this paper: https://www.researchgate.net/profile/Karolin_Luger/publication/12512310_Expression_and_purification_of_recombinant_histones_and_nucleosome_reconstitution/links/5526995e0cf2647b189d78a1.pdf)
Article Expression and Purification of Recombinant Histones and Nucl...
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