Analytical- and preparative-scale separation of molecular variants of alpha-fetoprotein by anion-exchange chromatography on Monobead resins.
A rapid and reliable purification procedure is described that is useful for both analytical detection and quantitative recovery of milligram amounts of individual molecular variants of mouse alpha-fetoprotein (AFP). The appropriate separation conditions were developed with an analytical-size Mono Q anion-exchange column linked to an automated Fast Protein Liquid Chromatography system. Effective separations of fetal-derived AFP variants was accomplished within 20 min under mild conditions with an L-histidine buffer. Employing the optimal separation conditions established on the Mono Q HR 5/5 column we upscaled the procedure by using a preparative Mono Q HR 16/10 column in order to obtain milligram quantities of each molecular variant of AFP. Seven distinct isomeric forms of AFP could be recovered on the preparative anion exchanger in a highly reproducible manner. Each of the seven protein peaks eluted from the Mono Q column were confirmed to be distinct isoforms of AFP by isoelectric focusing and Western blotting developed with monospecific anti-AFP antisera. This method in its scaled up version offers the benefit of providing milligram quantities of immunochemically pure AFP isomers for structure and function studies.
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
N S
NS
Van Oers
R
R
Boismenu
B L
BL
Cohen
R A
RA
Murgita
D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods*; D014644:Genetic Variation; D015151:Immunoblotting; D007525:Isoelectric Focusing; D051379:Mice; D012116:Resins, Plant; D000509:alpha-Fetoproteins / Q000032:analysis* / Q000235:genetics
D012116:Resins, Plant; D000509:alpha-Fetoproteins