title: Separation of phosphorylated histone H1 variants by high-performance capillary electrophoresis.
abstract: 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.
authors.affiliation: Institute of Medical Chemistry and Biochemistry, Innsbruck, Austria.
authors.forename: H
authors.initials: H
authors.lastname: Lindner
authors.forename: W
authors.initials: W
authors.lastname: Helliger
authors.forename: A
authors.initials: A
authors.lastname: Dirschlmayer
authors.forename: H
authors.initials: H
authors.lastname: Talasz
authors.forename: M
authors.initials: M
authors.lastname: Wurm
authors.forename: B
authors.initials: B
authors.lastname: Sarg
authors.forename: M
authors.initials: M
authors.lastname: Jaquemar
authors.forename: B
authors.initials: B
authors.lastname: Puschendorf
mesh_terms: 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
chemical_list: D006657:Histones; D000469:Alkaline Phosphatase