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.
Van Oers
N S
NS
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Boismenu
R
R
Cohen
B L
BL
Murgita
R A
RA
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