Quantification of Active Caspase 3 in Apoptotic Cells
| Autor: | Calvin F. Roff, David R. Cahill, Keith G. Godfrey, Mark J. Schendel, Amy M. Walz, Paul A. Saunders, Igor J. Woyno, Chad J. Borchert, Ryan T. Loegering, Frank Mortari, Alexander E. Kaluyzhny, Jeffrey A. Cooper, Michelle M. Roodell, Harvey Gaylord, Dorothy A. Schroeder, Richard A. Krzyzek, Monica Tsang, Amy L. Isaacson |
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| Rok vydání: | 2000 |
| Předmět: |
Silver Staining
Time Factors Immunoblotting Caspase 2 Biophysics Apoptosis Enzyme-Linked Immunosorbent Assay Caspase 3 Caspase 8 Biochemistry Caspase 7 Jurkat Cells Tumor Cells Cultured Humans Biotinylation fas Receptor Enzyme Inhibitors Caspase 10 Molecular Biology Horseradish Peroxidase Caspase Caspase-9 Dose-Response Relationship Drug biology U937 Cells Cell Biology Staurosporine Molecular biology Caspase 9 Recombinant Proteins Caspases biology.protein Streptavidin |
| Zdroj: | Analytical Biochemistry. 284:114-124 |
| ISSN: | 0003-2697 |
| Popis: | We describe an enzyme-linked immunosorbent assay (ELISA) for quantifying relative amounts of active caspase 3 in apoptotic cells. Covalent modification of caspase 3 active sites with a biotinylated inhibitor differentiates active from latent caspases. Capture on an ELISA plate with an antibody specific for caspase 3 makes the assay specific for caspase 3. Detection is with horseradish peroxidase (HRP)-conjugated streptavidin that binds to the biotinylated inhibitor covalently bound to caspase 3. Using the assay we detected 6.6 ng active caspase 3 per 10(6) apoptotic staurosporine-treated Jurkat cells. Specificity of the assay for caspase 3 was demonstrated by lack of signal with purified caspases 2, 7, 8, and 10 that were modified by a biotinylated inhibitor. Specificity was also demonstrated by lack of signal with apoptotic MCF-7 cells which do not express caspase 3. The ability to discriminate between active and latent caspase 3 was shown by Western blotting with HRP-streptavidin and anti-caspase 3. Although latent caspase 3 was captured it was not covalently modified with the biotinylated inhibitor. The basic principle of using a covalent inhibitor to identify active enzymes and an antibody to differentiate between enzymes with similar activities has potential for quantifying active members of many classes of enzymes. |
| Databáze: | OpenAIRE |
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