Rapid analysis of mitotic histone H1 phosphorylation by cationic disc electrophoresis at neutral pH in minigels.
A new method is described for analysis of histone H1 and other basic proteins by cationic disc electrophoresis in polyacrylamide gels at neutral pH. The multiphasic buffer (disc) system uses Na+ as leading ion, L-histidine as trailing ion, and Hepes as buffering counterion. These "Hepes/histidine gels" have three advantages over conventional acid-urea gels for studies of H1 phosphorylation and dephosphorylation: speed, convenience, and the need for only small amounts of cells or chromatin. Core histones and their acetylated forms can also be separated in gels containing 0.4% Triton X-100. The difference in electrophoretic mobility between mitotic (superphosphorylated) and interphase H1 from HeLa cells is approximately twice as great at neutral pH as at pH 4.5, making it possible to separate these two H1 forms rapidly and easily in Hepes/histidine "minigels" only 5-cm long. Total histones can be rapidly prepared by simply neutralizing 0.2 N HCl extracts, and the entire analysis, from harvesting cells to destaining gels, can be carried out in 1 day. The stacking effect of the disc system produces sharp bands and high resolution even with relatively dilute samples.
Paulson
J R
JR
Department of Chemistry, University of Wisconsin Oshkosh, 54901.
Mesner
P W
PW
Delrow
J J
JJ
Mahmoud
N N
NN
Ciesielski
W A
WA
D002843:Chromatin / Q000302:isolation & purification; D004589:Electrophoresis, Disc; D006531:HEPES; D006367:HeLa Cells; D006639:Histidine; D006657:Histones / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008938:Mitosis; D017830:Octoxynol; D010766:Phosphorylation; D011092:Polyethylene Glycols; D012964:Sodium / Q000737:chemistry; D013194:Staining and Labeling
D002843:Chromatin; D006657:Histones; D011092:Polyethylene Glycols; D006639:Histidine; D017830:Octoxynol; D012964:Sodium; D006531:HEPES