Analysis of nucleoproteins by direct injection of dissolved nuclei or chromosomes into a high-performance liquid chromatographic system.
This work describes the development of a method for fractionating the total nuclear proteins of Chinese hamster cells (line CHO) by high-performance liquid chromatography of whole nuclei or chromosomes. Nuclei or chromosomes were dissolved for 2 h in 6 M guanidine hydrochloride containing 0.2% trifluoroacetic acid (TFA). Residual particulates were removed by centrifugation, and a sample of the solution was passed through a Bio-Sil TSK guard column, followed by a Bio-Sil TSK 400 column, equilibrated with water containing 0.2% TFA. These columns fractionated the sample by adsorbing the DNA, passing the proteins near the exclusion limit, passing the guanidine at the inclusion limit, and passing an unidentified non-protein/non-DNA material after the inclusion limit. The protein peaks from the TSK columns were collected directly on a muBondapak CN reversed-phase column. The protein fractions were then eluted from the CN column with an acetonitrile gradient containing 0.2% TFA. Three other reversed-phase columns were examined for use with the TSK columns. The muBondapak C18 Radial-Pak column produced the best resolution of histone variants, while the Zorbax C8 column produced a better resolution of the non-histone proteins. The Nova-Pak C18 Radial-Pak column was found to be unsatisfactory for both classes of nucleoproteins.
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000032:analysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002875:Chromosomes / Q000032:analysis; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000302:isolation & purification; D006657:Histones / Q000302:isolation & purification; D009698:Nucleoproteins / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D014269:Trifluoroacetic Acid
D006657:Histones; D009698:Nucleoproteins; D004247:DNA; D014269:Trifluoroacetic Acid