Human perforin: rapid enrichment by immobilized metal affinity chromatography (IMAC) for whole cell cytotoxicity assays.
Perforin, a potent pore-forming protein, plays an important role in lymphocyte-mediated cytolysis causing necrosis or, when combined with the granzymes, apoptosis. The studies on perforin, although already extensive, have been hampered by the limited amount of material available from killer lymphocytes. Using a cell line that expresses high levels of human perforin, we describe a straightforward purification scheme that allows isolation of the lytic protein in approximately 8 hours. Perforin is enriched from the YT-INDY cell line by cavitation followed by differential centrifugation and ion metal affinity chromatography. In addition to demonstrating the lytic activity of human perforin toward various cell lines, we show that the conditions of the cytotoxicity assay influence both the kinetics and magnitude of perforin-mediated cytotoxicity.
Froelich
C J
CJ
Department of Medicine, Evanston Hospital, Northwestern University, Evanston, Illinois 60201, USA. granzyme@merle.acns.nwu.edu
Turbov
J
J
Hanna
W
W
D017209:Apoptosis / Q000502:physiology; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D003035:Cobalt; D003594:Cytoplasmic Granules; D003601:Cytotoxicity Tests, Immunologic; D006461:Hemolysis; D006801:Humans; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins
D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; D003035:Cobalt