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| Product Name | Caspase-6 Activity Colorimetric Assay Kit (VEID-pNA Substrate) |
| Catalog No. | CCAT-HMM-0064 |
| Description | A colorimetric assay for measuring caspase-6 activity in cell and tissue lysates using the chromogenic peptide substrate VEID-pNA (Ac-Val-Glu-Ile-Asp-p-nitroanilide). Active caspase-6 cleaves the substrate to release p-nitroaniline (pNA), which is quantified spectrophotometrically at 405 nm. Caspase-6 is an executioner caspase involved in lamin A cleavage during apoptosis and has been implicated in neurodegenerative disease pathways including Huntington's and Alzheimer's diseases. |
| Intended Use | Quantitative measurement of caspase-6 enzymatic activity; apoptosis pathway studies focusing on executioner caspase activation; neurodegenerative disease mechanism research; screening of caspase-6-specific inhibitors; validation of caspase activation cascades in cell death research. |
| Principle / Technology | Active caspase-6 specifically recognizes and cleaves the VEID tetrapeptide sequence, releasing p-nitroaniline (pNA) chromophore; absorbance of free pNA at 405 nm is directly proportional to caspase-6 enzymatic activity in the sample |
| Detection Method | Colorimetric; absorbance at 405 nm; 96-well plate format; endpoint or kinetic reading mode |
| Sample Type | Cell and tissue lysates; 100–200 µg total protein per assay recommended; prepare lysates using non-denaturing lysis buffer |
| Sensitivity / LOD | Detects caspase-6 activity from 500 cell equivalents; LOD 10 pmol pNA/min; linear range 0.01–2.0 OD at 405 nm |
| Specificity | Highly specific for caspase-6 (VEIDase activity); minimal cross-reactivity with caspase-3 (DEVDase) or caspase-8 (IETDase); use caspase-6 specific inhibitor Ac-VEID-CHO for signal specificity confirmation |
| Reaction Conditions / Protocol | Lyse cells in 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 200 µM VEID-pNA substrate; incubate at 37 °C for 1–4 hours; read absorbance at 405 nm |
| Components / Formulation | VEID-pNA substrate (lyophilized, 5 µmol), caspase-6 inhibitor Ac-VEID-CHO (lyophilized, 100 nmol), lysis buffer (10×), reaction buffer (2×), DTT (1 M), pNA calibration standard (10 mM in DMSO), dilution buffer, 96-well plate sealing film |
| Storage Conditions | Store at -20 °C; protect substrate and pNA standard from light; avoid repeated freeze-thaw cycles |
| Shelf Life | 12 months from manufacture date at -20 °C |
| Package Specifications | 100 assays, 200 assays, 500 assays |
| Product Form | Lyophilized substrate and inhibitor; liquid buffers; pNA standard in DMSO |
| Key Features | VEID substrate sequence optimized for caspase-6 specificity; pNA chromophore enables simple absorbance detection; includes specific inhibitor for signal validation; pNA calibration standard for absolute quantification; compatible with standard 96-well plate readers; detailed protocol with troubleshooting guide included |
| Purity | VEID-pNA purity ≥97% by HPLC; free pNA <0.2%; inhibitor purity ≥97%; no detectable protease contamination |
| 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 pNA reagents from light; DTT solution ships on cold pack |
| Expiration Date / Stability | 12 months at -20 °C; reconstituted pNA standard stable 1 month at -20 °C; reconstituted substrate stable 1 week at -20 °C; prepare fresh DTT working concentration for each assay |
| Regulatory / Compliance | For laboratory and research use only; RUO; manufactured under ISO 9001; pNA is a laboratory chemical — handle with gloves; not classified as dangerous goods |
| Compatibility | Compatible with mammalian cell lysates from human, mouse, and rat origin; lysis buffer is non-denaturing — avoid SDS, deoxycholate, and other ionic detergents that denature caspases; DMSO levels up to 2% in final reaction do not inhibit caspase-6 activity |
| Recommended Buffer System | Annexin binding buffer with calcium; cell lysis buffer with protease inhibitors for caspase assays |
| Application Notes / Precautions | For kinetic measurements, read absorbance every 10 minutes for 2–4 hours at 37 °C. Calculate activity from the initial linear portion of the curve. For inhibitor assays, pre-incubate lysate with Ac-VEID-CHO (1–5 µM final) for 15 minutes at 37 °C before adding substrate. Known caspase-6 inducers include staurosporine, etoposide, and TNF-α/cycloheximide combination. Always include substrate-only blank for background subtraction. |
| 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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