Separation of phosphorylated histone H1 variants by high-performance capillary electrophoresis.
High-performance capillary electrophoresis (HPCE) was used to separate successfully distinct phosphorylated derivatives of individual histone H1 variants. With an untreated capillary (50 cm x 75 microns I.D.) the electrophoresis was performed in about 15 min. Inconvenient interactions of these highly basic proteins with the capillary wall were eliminated by using 0.1 M sodium phosphate buffer (pH 2.0) containing 0.03% hydroxypropylmethylcellulose. Under these experimental conditions the histone H1 variants H1b and H1c obtained from mitotic enriched NIH 3T3 fibroblasts and isolated by reversed-phase high-performance liquid chromatography were clearly separated in their non-phosphorylated and different phosphorylated forms. This result was confirmed by acid-urea gel electrophoresis, comparison with non-phosphorylated histones H1b and H1c, isolated from quiescent NIH 3T3 cells, and incubation of multi-phosphorylated histone H1b with alkaline phosphatase and subsequent acid-urea and capillary electrophoresis. The results illustrate that the application of HPCE to the analysis of histone modifications provides a new alternative to traditional gel electrophoresis.
Lindner
H
H
Institute of Medical Chemistry and Biochemistry, Innsbruck, Austria.
Helliger
W
W
Dirschlmayer
A
A
Talasz
H
H
Wurm
M
M
Sarg
B
B
Jaquemar
M
M
Puschendorf
B
B
D000469:Alkaline Phosphatase; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006657:Histones / Q000032:analysis*; D051379:Mice; D010766:Phosphorylation
D006657:Histones; D000469:Alkaline Phosphatase