A combined recycling affinity column-filtration technique.
A new preparative purification technique for biological macromolecules is developed by combining the affinity column chromatography and the membrane filtration technique. A unique feature of the affinity column-filtration apparatus is that the filtrate from the filtration unit is recycled back to the inlet of the affinity column, thus decreasing considerably the total amount of ligands required for the affinity column. A second important feature is that the pump which connects the affinity column and the filtration unit is monitored by an infrared sensor device on the filtration cell; consequently, the flow of the column eluent into the filtration unit is controlled automatically by the solution height in the filtration cell. Highly homogeneous regulatory subunits of type I cAMP-dependent protein kinase from rabbit skeletal muscle are demonstrated to be obtainable by the new purification technique. Thus, this apparatus could have important applications in the purification of a wide range of biological macromolecules. A test for estimating affinity bound proteins is also discussed.
D000818:Animals; D002846:Chromatography, Affinity / Q000295:instrumentation*; D002848:Chromatography, DEAE-Cellulose; D005374:Filtration; D009132:Muscles / Q000201:enzymology; D011494:Protein Kinases / Q000302:isolation & purification; D011817:Rabbits
D011494:Protein Kinases