Separation of acetylated core histones by hydrophilic-interaction liquid chromatography.
Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of postranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phase high-performance liquid chromatography (RP-HPLC) we have successfully separated various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea-Triton gel electrophoresis. Our results demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to traditional separation techniques and will be useful in studying the biological function of histone acetylation.
Lindner
H
H
Institute of Medical Chemistry and Biochemistry, Innsbruck, Austria.
Sarg
B
B
Meraner
C
C
Helliger
W
W
D000107:Acetylation; D000143:Acids / Q000737:chemistry; D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000737:chemistry; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005622:Friend murine leukemia virus; D006657:Histones / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects; D004915:Leukemia, Erythroblastic, Acute; D017830:Octoxynol / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013499:Surface Properties; D014407:Tumor Cells, Cultured; D014508:Urea / Q000737:chemistry
D000143:Acids; D002087:Butyrates; D003902:Detergents; D006657:Histones; D020148:Butyric Acid; D014508:Urea; D017830:Octoxynol