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| Product Name | Caspase-1 Activity Assay Kit, Fluorometric, YVAD-AFC Substrate |
| Catalog No. | ETR-HMM-0090 |
| Description | Fluorometric caspase-1 (interleukin-1beta converting enzyme, ICE) activity assay kit using the specific fluorogenic peptide substrate Ac-YVAD-AFC (N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-trifluoromethylcoumarin). Upon cleavage by active caspase-1 at the Asp-AFC bond, the AFC fluorophore is released and emits blue-green fluorescence (Ex/Em = 400/505 nm) that is proportional to caspase-1 enzymatic activity. The assay is suitable for measuring caspase-1 activation in cell and tissue lysates following inflammasome activation stimuli (LPS + ATP, nigericin, alum crystals, etc.), as well as for purified caspase-1 enzyme studies. The kit includes Ac-YVAD-AFC substrate, a specific caspase-1 inhibitor (Ac-YVAD-CHO) for specificity confirmation, AFC calibration standard, and optimized lysis and assay buffers for consistent results. |
| Intended Use | Quantitative fluorometric measurement of caspase-1 protease activity in: cell lysates from inflammasome activation studies (NLRP3, NLRC4, AIM2, Pyrin inflammasomes); tissue homogenates from inflammatory disease models; purified recombinant caspase-1 enzyme characterization; caspase-1 inhibitor screening and IC50 determination; pyroptosis research and confirmation of caspase-1-dependent cell death pathway activation. |
| Principle / Technology | Active caspase-1 (a heterotetramer of two p20 and two p10 subunits) specifically recognizes and cleaves the tetrapeptide sequence YVAD (Tyr-Val-Ala-Asp) between Asp and the AFC leaving group. The released AFC (7-amino-4-trifluoromethylcoumarin) is fluorescent (Ex/Em = 400/505 nm) while the intact Ac-YVAD-AFC substrate is minimally fluorescent. The fluorescence increase rate (delta RFU/min) is proportional to caspase-1 activity. Specificity is confirmed by parallel reactions containing the caspase-1 inhibitor Ac-YVAD-CHO (competitive, reversible) or Ac-YVAD-CMK (irreversible). |
| Detection Method | Harvest cells and lyse in caspase-1 lysis buffer (10 min on ice); centrifuge 10,000 x g for 10 min at 4 C; transfer supernatant; determine protein concentration (BCA or Bradford); incubate 50-200 ug lysate protein with Ac-YVAD-AFC (50 uM final) in assay buffer in black 96-well plate at 37 C; measure fluorescence (Ex/Em 400/505 nm) kinetically every 1-5 min for 30-120 min; calculate activity from the linear portion of the progress curve; confirm specificity with Ac-YVAD-CHO (1 uM) inhibition. |
| Sample Type | Cell lysates from: THP-1 macrophages, J774A.1 macrophages, bone marrow-derived macrophages (BMDMs), dendritic cells, and other inflammasome-competent cell types after stimulation; tissue homogenates (spleen, liver, lung); purified recombinant caspase-1 (active form). |
| Performance Range / Specifications | Detection range: 0.1-100 pmol AFC per well; linear range: 0.1-5,000 RFU (fluorometer-dependent); assay duration: 30-120 min kinetic at 37 C. |
| Sensitivity / LOD | Detection of as little as 0.5 uU caspase-1 activity per well (1 U = 1 pmol AFC released per minute); corresponds to caspase-1 activation in approximately 5x10^4 THP-1 cells after LPS + ATP stimulation. |
| Specificity | Ac-YVAD-AFC is a specific caspase-1 substrate (kcat/Km ~1x10^5 M^-1 s^-1); cross-reactivity with caspase-4/caspase-5 (also inflammatory caspases) is approximately 10-20% (WEHD preferred substrate); caspase-3 and caspase-7 show <1% activity with YVAD sequence. Ac-YVAD-CHO inhibitor is specific for caspase-1 (IC50 ~0.5-2 nM) with >100-fold selectivity over caspase-3. |
| Reaction Conditions / Protocol | Assay conditions: 50-200 ug cell lysate, 50 uM Ac-YVAD-AFC, 37 C, pH 7.2-7.4; kinetic monitoring at Ex/Em 400/505 nm; typical reaction time 30-120 min; include inhibitor control with 1 uM Ac-YVAD-CHO. |
| Components / Formulation | Ac-YVAD-AFC Substrate (10 mM in DMSO, 50 uL), Ac-YVAD-CHO Inhibitor (1 mM in DMSO, 10 uL), AFC Calibration Standard (1 mM in DMSO, 10 uL), Lysis Buffer (5x, 25 mL), Assay Buffer (2x, 25 mL), DTT (1 M, 0.5 mL), Black 96-Well Plate, Protocol. |
| Storage Conditions | Ac-YVAD-AFC at -20 C desiccated and protected from light; Ac-YVAD-CHO at -20 C; AFC standard at -20 C protected from light; Lysis and Assay buffers at 2-8 C; DTT at -20 C. |
| Shelf Life | 12 months from date of manufacture; peptide substrates stable at -20 C for 24 months lyophilized, 6 months in DMSO solution. |
| Package Specifications | 1 kit (sufficient for 200 tests in 100 uL assay volume). |
| Product Form | Liquid peptide substrate solution (DMSO); liquid inhibitor (DMSO); liquid AFC standard (DMSO); liquid buffers; DTT solution. |
| Quality Control | Each lot tested for: substrate purity >=95% by HPLC; AFC release linearity with caspase-1 standard (R2 >0.99 over 0-100 pmol AFC/min); inhibitor efficiency: >90% inhibition of 1 U caspase-1 with 1 uM Ac-YVAD-CHO; background fluorescence (substrate auto-hydrolysis) change <0.5 RFU/min at 37 C. |
| Key Features | Fluorometric YVAD-AFC substrate; caspase-1 specific; inhibitor included for specificity control; AFC standard for absolute quantification; optimized for inflammasome research; kinetic and endpoint compatible; black 96-well plate included. |
| Purity | Ac-YVAD-AFC >=95% by HPLC; AFC standard >=99% by HPLC; no fluorescent contaminants in substrate; DMSO: anhydrous grade. |
| Concentration | Ac-YVAD-AFC: 10 mM in DMSO (working concentration 50 uM final); Ac-YVAD-CHO: 1 mM in DMSO (working concentration 0.1-1 uM); AFC standard: 1 mM in DMSO for calibration curve 0-10 uM. |
| Activity / Unit Definition | 1 caspase-1 unit = 1 pmol AFC released per minute at 37 C, pH 7.2; molecular activity (kcat) of caspase-1 for Ac-YVAD-AFC approximately 0.5-1 s^-1 at 37 C. |
| Molecular Weight | Ac-YVAD-AFC: 651.6 g/mol; Ac-YVAD-CHO: 574.6 g/mol; AFC: 229.2 g/mol; active caspase-1 (p20/p10)2 tetramer: ~60 kDa. |
| Source / Origin | Peptide substrates and inhibitors: synthetic (solid-phase peptide synthesis); AFC: synthetic; all buffers: molecular biology grade; DMSO: anhydrous grade; no animal-derived components. |
| pH Range / Optimal pH | Optimal pH 7.2-7.4 (caspase-1 activity); lysis buffer pH 7.5; assay buffer pH 7.2; AFC fluorescence pH dependent (maximal above pH 7.0). |
| Shipping Conditions | Cold pack (2-8 C); peptide substrates and inhibitor stable at ambient for 3 days (recommend cold pack for extended transit). |
| Expiration Date / Stability | 12 months at recommended conditions; DMSO solutions of peptides stable at -20 C for 6 months; AFC standard stable at -20 C for 12 months; buffers stable at 2-8 C for 12 months. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Cell lysis buffer: non-denaturing, compatible with downstream protein assay (BCA or Bradford). Do not use RIPA or other denaturing buffers as they inactivate caspase-1. DTT (1-10 mM) is required in assay buffer to maintain active-site cysteine in reduced state. Fresh DTT should be added to assay buffer before each use. Avoid freeze-thaw of cell lysates (measure caspase-1 activity immediately after lysis or store lysates at -80 C). Compatible with black 96-well and 384-well plates; white plates may produce higher signal. |
| Recommended Buffer System | Lysis Buffer: 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% CHAPS, protease inhibitor cocktail (add fresh); Assay Buffer: 50 mM HEPES pH 7.2, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT (add fresh), 1 mM EDTA. |
| Application Notes / Precautions | Include the following controls: (1) Substrate blank: assay buffer + substrate only (measure auto-hydrolysis rate); (2) Inhibitor control: sample + 1 uM Ac-YVAD-CHO (should reduce activity by >85% for caspase-1-specific signal); (3) Positive control: purified active caspase-1 or lysate from LPS+ATP-treated THP-1 cells; (4) Unstimulated cell control: lysate from untreated cells (baseline caspase-1 activity). Pre-warm plate and assay buffer to 37 C before adding substrate. DTT must be added fresh to assay buffer (1 mM working, from 1 M stock) to maintain reducing conditions. For inflammasome studies, typical stimuli: LPS priming (1 ug/mL, 3-4 h) followed by ATP (5 mM, 45 min), nigericin (10 uM, 45 min), or alum (300 ug/mL, 6 h). |
| Batch-to-Batch Consistency | Caspase-1 standard curve linearity R2 >0.99; inhibitor efficiency >90% at 1 uM; substrate auto-hydrolysis <0.5 RFU/min; AFC standard curve linearity R2 >0.999. |
For research use only, not for clinical use.
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