دورية أكاديمية
Quantification of active caspase 3 in apoptotic cells.
| العنوان: | Quantification of active caspase 3 in apoptotic cells. |
|---|---|
| المؤلفون: | Saunders PA; R & D Systems, Inc., Minneapolis, Minnesota 55413, USA., Cooper JA, Roodell MM, Schroeder DA, Borchert CJ, Isaacson AL, Schendel MJ, Godfrey KG, Cahill DR, Walz AM, Loegering RT, Gaylord H, Woyno IJ, Kaluyzhny AE, Krzyzek RA, Mortari F, Tsang M, Roff CF |
| المصدر: | Analytical biochemistry [Anal Biochem] 2000 Aug 15; Vol. 284 (1), pp. 114-24. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Elsevier Country of Publication: United States NLM ID: 0370535 Publication Model: Print Cited Medium: Print ISSN: 0003-2697 (Print) Linking ISSN: 00032697 NLM ISO Abbreviation: Anal Biochem Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: <2000- > : San Diego, CA : Elsevier Original Publication: Orlando Fl : Academic Press |
| مواضيع طبية MeSH: | Apoptosis*, Caspases/*biosynthesis, Biotinylation ; Caspase 10 ; Caspase 2 ; Caspase 3 ; Caspase 7 ; Caspase 8 ; Caspase 9 ; Caspases/metabolism ; Caspases/pharmacology ; Dose-Response Relationship, Drug ; Enzyme Inhibitors/pharmacology ; Enzyme-Linked Immunosorbent Assay ; Horseradish Peroxidase/metabolism ; Humans ; Immunoblotting ; Jurkat Cells ; Recombinant Proteins/metabolism ; Silver Staining ; Staurosporine/pharmacology ; Streptavidin/metabolism ; Time Factors ; Tumor Cells, Cultured ; U937 Cells ; fas Receptor/immunology |
| مستخلص: | 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. (Copyright 2000 Academic Press.) |
| المشرفين على المادة: | 0 (Enzyme Inhibitors) 0 (Recombinant Proteins) 0 (fas Receptor) 9013-20-1 (Streptavidin) EC 1.11.1.- (Horseradish Peroxidase) EC 3.4.22.- (CASP3 protein, human) EC 3.4.22.- (CASP7 protein, human) EC 3.4.22.- (CASP8 protein, human) EC 3.4.22.- (CASP9 protein, human) EC 3.4.22.- (Caspase 10) EC 3.4.22.- (Caspase 2) EC 3.4.22.- (Caspase 3) EC 3.4.22.- (Caspase 7) EC 3.4.22.- (Caspase 8) EC 3.4.22.- (Caspase 9) EC 3.4.22.- (Caspases) EC 3.4.22.63 (CASP10 protein, human) H88EPA0A3N (Staurosporine) |
| تواريخ الأحداث: | Date Created: 20000810 Date Completed: 20001002 Latest Revision: 20171116 |
| رمز التحديث: | 20231215 |
| DOI: | 10.1006/abio.2000.4690 |
| PMID: | 10933864 |
| قاعدة البيانات: | MEDLINE |
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