A simplified and efficient procedure for the purification of apolipoprotein AI from human serum high-density lipoprotein-3 by preparative isoelectric focussing on polyacrylamide gel beads.
An improved method is described for the purification of milligram amounts of apolipoprotein AI from serum apo-HDL3 by isoelectric focussing on polyacrylamide gel beads. The procedure involves a single focussing over a narrow (1.3 unit) pH gradient, and permits isolation of apo-AI of exceptional purity and in high yield (75% recovery of HDL3 protein, ca. 50% corresponding to pure apo-AI). The electrophoretic mobility, pI values, molecular weight, antigenicity and amino acid composition of such apo-AI were indistinguishable from those reported in the literature. A rabbit antiserum to apo-AI isolated by focussing exhibited similar immunological reactivity to one prepared from an antigen isolated by gel filtration chromatography; moreover, apo-AI purified by the respective procedures reacted identically with both antisera. We conclude that isoelectric focussing on a support of polyacrylamide gel beads (as Bio-Gel P60) presents certain advantages for the isolation of highly purified apo-AI over both conventional chromatographic procedures and isoelectric focussing on a Sephadex support.
D000180:Acrylic Resins; D000328:Adult; D000596:Amino Acids / Q000032:analysis; D016632:Apolipoprotein A-I; D001053:Apolipoproteins / Q000276:immunology / Q000302:isolation & purification*; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007525:Isoelectric Focusing / Q000379:methods*; D008075:Lipoproteins, HDL / Q000097:blood*; D008970:Molecular Weight
D000180:Acrylic Resins; D000596:Amino Acids; D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D000939:Epitopes; D008075:Lipoproteins, HDL; C016680:polyacrylamide gels