Salt gradient dialysis reconstitution of nucleosomes.

Abstract

INTRODUCTIONIn the salt gradient dialysis method, purified core histones are incubated with a DNA template in a buffer containing a high concentration of NaCl. As the salt is slowly dialyzed away, nucleosomes spontaneously assemble on the DNA, and their translational positioning along the DNA is directed by the DNA sequence. In the absence of nucleosome-positioning elements (e.g., 5S rDNA genes), the nucleosomes can adopt a closely packed nonphysiological structure with little space between the nucleosomes. Removal of remaining free histones, as well as templates with closely packed nucleosomes, can be achieved by fractionation over sucrose gradients. The chromatin assembled in these reactions can then be analyzed using micrococcal nuclease digestion. Salt dialysis reconstitutions are easy to perform, but they are time-consuming because of multiple changes of the dialysis buffer. The reconstitution method presented here takes a total of 1.5-2 d to complete.