Yeast Histone Purification
Yeast recombinant histones are purified from a pET-based E. coli expression vector. (For human histone purification, see Human Histone Purification and Expression and Purification of Human Histone 3x FLAG tagged hH2A. For reconstituting octamers onto 601 DNA, see Nucleosome Reconstitution.)
Contents
Section titled “Contents”1. Before You Begin
Section titled “1. Before You Begin”Urea and carbamylation of proteins: With heat and time, urea can break down into ammonium cyanate. Isocyanic acid will attack primary amines in proteins (Arg, Lys, N-terminus) to make a carbamylated protein. This interferes with histone structure and function. Many early or sloppy in vitro studies have suffered from this problem for decades. Source: http://www.ionsource.com/Card/carbam/carbam.htm
2. Purification
Section titled “2. Purification”- yeast H2A
- yeast H2B
- yeast H3
- yeast H4
3. Nucleosome Reconstitution
Section titled “3. Nucleosome Reconstitution”For now, read the methods paper (Methods Mol Biol vol. 778, p. 243).
4. Salt dialysis onto DNA
Section titled “4. Salt dialysis onto DNA”For now, read the methods paper (Methods Mol Biol vol. 778, p. 243).