Purification of colony-stimulating factor by affinity chromatography.
Studies were undertaken to determine whether L-cell-derived colony-stimulating factor (CSF) could be purified by a single step affinity chromatographic technique. A quantity of 100 X 10(6) units of purified anti-CSF was coupled to cyanogen bromide activated Sepharose 4B; colony assays revealed complete binding of the antibodies to the gel. Three 10-liter pools of serum-free L-cell CSF were concentrated by ultrafiltration, applied to the gel, and eluted with a low pH, high molarity buffer. Recovery of CSF ranged from 68%-100% with greater than 1000-fold decreases in protein content. Specific activity of the purified CSF ranged from 2.8 to 5.9 X 10(7) U of CSF/mg protein. Following iodination, each purified pool of CSF revealed a major 63,000-dalton peak of radioactivity that comigrated with CSF activity in SDS-acrylamide gels. In addition, several smaller peaks of 50,000 and 40,000 molecular weight were also detected. Approximately two-thirds of the purified CSF was adherent to concanavalin-A with elution by a competing sugar. Electrophoretic mobility was retarded by incubation with neuraminidase. These chromatographic studies confirm the observation that CSF is a glycoprotein but also suggest variable degrees of glycosylation of the molecule. This chromatographic technique should prove useful in the rapid purification of large quantities of CSF for physiologic and biochemical characterization.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D007739:L Cells / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008970:Molecular Weight; D011817:Rabbits
D000906:Antibodies; D003115:Colony-Stimulating Factors