Procedures for the purification of interleukin 3 to homogeneity.
A procedure is described for the routine purification of IL 3 to homogeneity from WEHI-3-conditioned media. The techniques employed include ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite, and G-75 Sephadex column chromatography. The last step in purification involves chromatography on C18 hydrophobic supports in RP-HPLC systems, which results in the coelution of a protein peak and IL 3 activity. This purification sequence results in approximately a 1,000,000-fold purification from the initial starting material with yields of 5 to 10% of the initial activity. typically, 150 liters of conditioned media yields 2 to 10 micrograms of IL 3. The purified material was homogeneous by SDS-PAGE analysis and had an apparent m.w. of 28,000. Purified IL 3 had a specific activity of approximately 0.05 ng/unit of activity. Additional criteria used to establish the relationship of the 28,000-dalton protein to IL 3 include the ability of an antiserum against IL 3 to concomitantly immunoprecipitate the iodinated protein and to inhibit its biologic activity as well as the ability of the iodinated protein to bind specifically to cell lines known to require IL 3 for growth.
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D007375:Interleukin-1 / Q000302:isolation & purification*; D007377:Interleukin-3; D008222:Lymphokines / Q000302:isolation & purification*; D051379:Mice; D008970:Molecular Weight; D013601:T-Lymphocytes / Q000276:immunology*
D007375:Interleukin-1; D007377:Interleukin-3; D008222:Lymphokines