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| Product Name | Caspase-1 Activity Fluorometric Assay Kit (YVAD-AFC Substrate) |
| Catalog No. | CCAT-HMM-0061 |
| Description | A fluorometric assay for quantitative measurement of caspase-1 (ICE, interleukin-1β converting enzyme) activity in cell lysates. The kit uses the caspase-1-specific fluorogenic peptide substrate YVAD-AFC (Ac-Tyr-Val-Ala-Asp-7-amino-4-trifluoromethylcoumarin), which upon cleavage by active caspase-1 releases fluorescent AFC that is quantified at 400/505 nm. This assay is critical for studying inflammasome activation, pyroptosis, and inflammatory cell death pathways. |
| Intended Use | Quantitative measurement of caspase-1 enzymatic activity in cell and tissue lysates; inflammasome activation studies (NLRP3, NLRC4, AIM2); pyroptosis pathway analysis; screening of caspase-1 inhibitors and activators; bacterial infection-mediated inflammatory response studies. |
| Principle / Technology | Active caspase-1 specifically cleaves the tetrapeptide sequence YVAD (Tyr-Val-Ala-Asp) in the fluorogenic substrate, releasing free AFC (7-amino-4-trifluoromethylcoumarin) which fluoresces at 505 nm when excited at 400 nm; fluorescence increase over time is proportional to caspase-1 activity |
| Detection Method | Fluorometric; excitation 380–400 nm, emission 490–510 nm; kinetic or endpoint mode; 96-well or 384-well plate format |
| Sample Type | Cell lysates from mammalian cells; requires 50–200 µg total protein per assay; use non-denaturing lysis buffer |
| Sensitivity / LOD | Detects caspase-1 activity from 500 cell equivalents; LOD 0.1 pmol AFC/min; linear response 0.1–50 pmol AFC |
| Specificity | Highly specific for caspase-1 and caspase-4; minimal cross-reactivity with caspase-3 (DEVDase activity); use caspase-1 specific inhibitor (Ac-YVAD-CHO or VX-765) for validation of signal specificity |
| Reaction Conditions / Protocol | Lyse cells in provided lysis buffer (10 minutes on ice); centrifuge at 12,000 × g for 10 minutes at 4 °C; transfer 50 µL supernatant to assay plate; add 50 µL 2× reaction buffer containing 50 µM YVAD-AFC substrate; incubate at 37 °C for 1–2 hours; read fluorescence; for inhibitor validation, pre-incubate lysate with inhibitor for 15 minutes before adding substrate |
| Components / Formulation | YVAD-AFC substrate (lyophilized, 1 µmol), caspase-1 inhibitor Ac-YVAD-CHO (lyophilized, 50 nmol), lysis buffer (10×), reaction buffer (2×), DTT (1 M), AFC calibration standard (1 mM), dilution buffer |
| Storage Conditions | Store at -20 °C; protect substrate and AFC standard from light; DTT store at -20 °C in single-use aliquots |
| Shelf Life | 12 months from manufacture date at -20 °C |
| Package Specifications | 100 assays, 200 assays, 500 assays in 96-well plate format |
| Product Form | Lyophilized substrate and inhibitor; liquid buffers |
| Key Features | Highly specific YVAD tetrapeptide substrate; includes specific caspase-1 inhibitor for signal validation; AFC standard for absolute quantification of enzyme activity; simple lysis-to-assay workflow in under 3 hours; DTT included for maintaining enzyme activity; compatible with inflammasome activators (LPS + ATP, nigericin, alum, MSU crystals) |
| Purity | YVAD-AFC purity ≥98% by HPLC; free AFC <0.1%; caspase-1 inhibitor purity ≥97% |
| Concentration | As specified per kit; sufficient for stated number of tests |
| Activity / Unit Definition | Apoptosis induction confirmed by positive control treatment producing ≥5-fold signal increase |
| Molecular Weight | Varies by dye and enzyme component |
| Source / Origin | Recombinant annexin proteins; synthetic fluorophore conjugates; chromogenic/fluorogenic peptide substrates |
| pH Range / Optimal pH | Binding/washing buffer pH 7.2–7.4 |
| Shipping Conditions | Cold pack -20 °C; protect substrate from light; avoid freeze-thaw of DTT solution |
| Expiration Date / Stability | 12 months at -20 °C; reconstituted AFC standard stable 1 month at -20 °C; reconstituted substrate stable 1 week at -20 °C; prepare fresh DTT working solution for each assay |
| Regulatory / Compliance | For laboratory and research use only; RUO; manufactured under ISO 9001; reagents not classified as dangerous goods |
| Compatibility | Compatible with mammalian cell lysates from human, mouse, and rat origin; non-denaturing lysis buffer preserves enzyme activity; avoid SDS and other denaturing detergents in lysis buffer; common caspase inhibitors (Z-VAD-FMK) also inhibit caspase-1 — use specific inhibitors for validation |
| Recommended Buffer System | Annexin binding buffer with calcium; cell lysis buffer with protease inhibitors for caspase assays |
| Application Notes / Precautions | For inflammasome activation, prime cells with LPS (100 ng/mL, 3–4 hours) then activate with ATP (5 mM, 30–45 minutes) or nigericin (10 µM, 30–45 minutes). For caspase-1 specificity confirmation, pre-incubate lysates with Ac-YVAD-CHO (1 µM final) for 15 minutes at 37 °C. Normalize activity to protein content (BCA assay). For kinetic measurements, read fluorescence every 5 minutes for 60–120 minutes; the initial linear rate is used for calculation. |
| Batch-to-Batch Consistency | Positive control signal within ±20% of reference lot; staining pattern consistent |
For research use only, not for clinical use.
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