Biotin-ZVKD-fmk modified natural and biotin-D-fmk modified recombinant active Caspase-3. This assay also detects mouse active Caspase-3.
< 0.5% cross-reactivity observed with available related molecules.Cross-species reactivity observed with 1 or more species tested.
No significant interference observed with available related molecules.
The Quantikine Human active Caspase-3 Immunoassay is a 3.5 hour solid phase ELISA designed to measure human active caspase-3 in cell extracts. It contains biotinylated-inhibitor derivatized recombinant caspase-3 and a monoclonal antibody raised against caspase-3. Results obtained using natural active caspase-3 covalently bound to biotin-ZVKD-fmk inhibitor in apoptotic cell extracts showed linear curves that were parallel to the Quantikine kit standard. These results indicate that this kit can be used to determine relative mass values for natural active caspase-3.
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.
The recovery of active caspase-3 spiked to three levels throughout the range of the assay in various matrices was evaluated.
Average % Recovery
Calibrator Diluent RD5-20 (1X) (n=4)
Extraction Buffer (1X) (n=4)
Extraction Buffer (1X) (n=4)
Extracts from Jurkat Cells Treated w/Staurosporine (n=4)
To assess the linearity of the assay, five or more samples containing and/or spiked with various concentrations of active caspase-3 in each matrix were diluted with Calibrator Diluent and then assayed. Results from typical sample dilutions are
Preparation and Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Caspases are a family of cytosolic aspartate-specific cysteine proteases involved in the initiation and execution of apoptosis. They are expressed as latent zymogens and are activated by an autoproteolytic mechanism or by processing by other proteases (frequently other caspases). Human caspases can be subdivided into three functional groups: cytokine activation (caspase-1, -4, -5, and -13), apoptosis initiation (caspase-2, -8, -9, -and -10), and apoptosis execution (caspase-3, -6, and -7).
Caspases are regulated by a variety of stimili, including APAF1, CFLAR/FLIP, NOL3/ARC, and members of the inhibitor of apoptosis (IAP) family such as BIRC1/NAIP, BIRC2/cIAP-1, BIRC3/cIAP-2, BIRC4/XIAP, BIRC5/Survivin, and BIRC7/Livin. IAP activity is modulated by DIABLO/SMAC or PRSS25/HTRA2/Omi. Cell-permeable and irreversible peptide inhibitors are also available for different caspases.
Refer to the product for complete assay procedure.
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
Prepare all reagents, standard dilutions, and samples as directed in the product insert.
Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
100 µL Standard, Control, or Sample
Add 100 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.
Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.
100 µL Conjugate
Add 100 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 1 hour.
Aspirate and wash 5 times.
100 µL Substrate Solution
Add 100 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes. PROTECT FROM LIGHT.
100 µL Stop Solution
Add 100 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.
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