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| Product Name | Caspase-3 Colorimetric Activity Assay Kit |
| Catalog No. | CCAT-HMM-0045 |
| Description | A colorimetric enzymatic assay kit that measures caspase-3 (CPP32/apopain) activity in cell and tissue lysates using the chromogenic substrate Ac-DEVD-pNA. Active caspase-3 specifically cleaves the DEVD tetrapeptide sequence to release free p-nitroaniline (pNA), whose absorbance is measured at 405 nm. |
| Intended Use | Quantitative measurement of caspase-3 protease activity as a hallmark indicator of apoptosis execution in cells treated with pro-apoptotic stimuli, for drug mechanism studies and compound screening. |
| Principle / Technology | Caspase-3 (also known as CPP32) is a key executioner caspase that is activated by initiator caspases (caspase-8 and caspase-9) during apoptosis; the chromogenic tetrapeptide substrate Ac-DEVD-pNA contains the caspase-3 recognition motif Asp-Glu-Val-Asp linked to p-nitroaniline; cleavage releases free pNA whose absorbance at 405 nm is proportional to caspase-3 activity. |
| Detection Method | Colorimetric absorbance at 405 nm measured kinetically or as an endpoint using a microplate spectrophotometer |
| Sample Type | Cell lysates from adherent or suspension mammalian cells; tissue homogenates from organs undergoing apoptosis; purified recombinant caspase-3 |
| Performance Range / Specifications | Specific activity measurable in lysates from as few as 1 × 10^5 apoptotic cells; linear pNA release over 1–4 hour incubation depending on caspase-3 concentration |
| Sensitivity / LOD | Detection of caspase-3 activity in samples containing ≥2 × 10^5 apoptotic cells in a typical 6-well format |
| Specificity | Ac-DEVD-pNA is a caspase-3 preferred substrate; cross-reactivity with caspase-7 is approximately 5% under standard assay conditions; minimal hydrolysis by caspase-2, -6, -8, -9, or -10; background from non-caspase proteases is negligible |
| Reaction Conditions / Protocol | Prepare cell lysate in lysis buffer (10^6 cells per 50 μL buffer), incubate on ice for 10 minutes, centrifuge at 10,000 × g for 1 minute; transfer supernatant to microplate; add 2× Reaction Buffer containing DTT; add Ac-DEVD-pNA substrate (200 μM final); incubate at 37°C for 1–2 hours; read absorbance at 405 nm; compare to pNA standard curve for absolute quantification |
| Components / Formulation | Cell Lysis Buffer (HEPES with CHAPS, DTT, and protease inhibitors), 2× Reaction Buffer (HEPES, NaCl, EDTA, DTT), Ac-DEVD-pNA substrate (10 mM stock in DMSO), pNA calibration standard, caspase-3 inhibitor Ac-DEVD-CHO for specificity control |
| Storage Conditions | -20°C for all components protected from light; substrate stock sensitive to moisture; lysis buffer supplemented with DTT fresh before use |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 100 assays, 200 assays (96-well microplate format) |
| Product Form | Concentrated lyophilized or liquid reagents requiring reconstitution or dilution |
| Quality Control | Each lot tested with recombinant active caspase-3 to determine specific activity (nmol pNA/min/unit enzyme); inhibitor efficiency verified >90% reduction in signal with Ac-DEVD-CHO; pNA standard linearity R2 ≥0.99 |
| Key Features | Colorimetric detection compatible with standard absorbance plate readers; quantitative pNA standard curve enables absolute activity determination; inhibitor Ac-DEVD-CHO included for specificity confirmation; adaptable to both endpoint and kinetic measurement modes |
For research use only, not for clinical use.
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