Human perforin: rapid enrichment by immobilized metal affinity chromatography (IMAC) for whole cell cytotoxicity assays
| Autor: | Jane Turbov, Christopher J. Froelich, W. L. Hanna |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Pore Forming Cytotoxic Proteins
Biophysics chemical and pharmacologic phenomena Apoptosis Cytoplasmic Granules Biochemistry Hemolysis Chromatography Affinity Cell Line Humans Cytotoxicity Molecular Biology Differential centrifugation Membrane Glycoproteins biology Perforin hemic and immune systems Cell Biology Cobalt Cytotoxicity Tests Immunologic Molecular biology Cytolysis Granzyme Lytic cycle Cell culture biology.protein |
| Zdroj: | Biochemical and biophysical research communications. 229(1) |
| ISSN: | 0006-291X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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