Capillary electrophoresis of histone H1 variants at neutral pH in dynamically modified fused- silica tubing.
Existing methods for the analysis of histone H1 by capillary electrophoresis (CE) employ acidic buffers (pH <3.0) to suppress silanol ionization and minimize the loss of these extremely basic proteins by adsorption to capillary walls. Here we describe the use of Polybrene (PB) as a dynamic modification reagent in a simple procedure that facilitates the analysis of chicken H1 at neutral pH. PB is adsorbed to the inner surfaces of capillaries to render them cationic prior to use and a low concentration of PB is included in the electrolyte to replenish the coating during use. Inclusion of ethylenediaminetetraacetic acid (EDTA) in the electrolyte results in the assembly of a dynamic cation-exchange layer upon the immobilized PB that influences the relative mobilities of H1 variants. The six nonallelic variants of H1 known in this species as well as certain allelic variants are resolved. Because the procedure is effective in preventing the adsorption of proteins as basic as H1 at neutral pH, this strategy should facilitate CE analyses of many basic proteins under conditions that maintain their native conformation.
Centre for Research in Neurodegenerative Diseases, University of Toronto, ON, Canada. cam8y@virginia.edu
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McLachlan
D019342:Acetic Acid; D000818:Animals; D002021:Buffers; D002645:Chickens; D002852:Chromatography, Ion Exchange / Q000379:methods; D004573:Electrolytes; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006583:Hexadimethrine Bromide; D006657:Histones / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D020033:Protein Isoforms / Q000032:analysis; D012822:Silicon Dioxide; D012967:Sodium Dodecyl Sulfate; D014508:Urea
D002021:Buffers; D004573:Electrolytes; D006657:Histones; D007202:Indicators and Reagents; D020033:Protein Isoforms; D012967:Sodium Dodecyl Sulfate; D006583:Hexadimethrine Bromide; D012822:Silicon Dioxide; D014508:Urea; D019342:Acetic Acid