Caspase 3 Assay Kit, Fluorimetric
SIGMA/CASP3F
Synonym: Caspase-3 Activity Fluorometric Kit
EC Number: 247-454-8
Product Type: Chemical
Caspase 3 activity in lysates of Jurkat cells induced with staursporine Jurkat cells were induced with 1 μg/ml staurosporine for 0, 1, 2, and 3 hours. At the end of the time, the cells were collected and lysed using 500 μl lysis buffer per 107 cells. Caspase 3 activity per 5 μl of either cell lysate or 0.5 μg/ml caspase 3 positive control solution (C) was measured fluorometrically in 96 well multiwell plates. The reaction volume was 200 μl containing 16.6 μM Ac-DEVD-AMC in 1X assay buffer. The assay was performed at room temperature, and readings were taken every 15 minutes, starting at 25 minutes.
Caspase 3 Activity: Jurkat cells treated with Zapotin (Cat. No. Z4652), Siomycin A (Cat. No. S6076) and Ikarugamycin (Cat. No. SML0188), for the indicated times and concentrations underwent apoptosis. Cells were washed and analyzed for Caspase 3 activity using the Caspase 3 Assay Kit, Fluorimetric (Cat. No. CASP3F) following the kit technical bulletin. Results are presented as fluorimetric units per mg protein. As observed, apoptosis kinetics diverted between the various antibiotics, as Ikarugamycin reached its maximum activity at 24 hr post treatment with 5 μM, while Siomycin A maximal induction was detected 8 hr post treatment with 5 μM. However treatment with 50 μM Zapotin for 8 hr seemed to induce Caspase-3 activity more efficiently than treatment with the other antibiotics tested.
| Quality Level | 200  |
| shipped in | dry ice |
| storage temp. | −20°C |
| usage | sufficient for 1,000 multiwell tests |
| sufficient for 100 standard tests (2 ml) |
| Application: | The Caspase 3 Fluorimetric Assay Kit is designed for the detection and measurement of caspase 3 activity in cell extracts or purified preparations of caspase 3. |
| General description: | The Caspase 3 Fluorometric Assay Kit is based on the hydrolysis of acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin (Ac-DEVD-AMC) by caspase 3, resulting in the release of the fluorescent 7-amino-4-methylcoumarin (AMC). The excitation and emission wavelengths of AMC are 360 nm and 460 nm respectively. The concentration of the AMC released can be calculated from a calibration curve prepared with AMC standards (AMC standard included with the kit). |
| Other Notes: | One unit of caspase 3 will cleave 1.0 μmol of Ac-DEVD per min at pH 7.4 at 25 °C. |
| Symbol |  GHS07 |
| Signal word | Warning |
| Hazard statements | H315 - H319 - H335 |
| Precautionary statements | P261 - P305 + P351 + P338 |
| Hazard Codes | Xi |
| Risk Statements | 36/37/38 |
| Safety Statements | 26-36 |
| RIDADR | NONH for all modes of transport |
| Storage Temp. | −20°C |
| UNSPSC | 12161503 |
| Components | Ac-DEVD-AMC substrate 2.5 mg; Ac-DEVD-CHO inhibitor .5 mg; 7-Amino-4-methylcoumarin standard 1 mg; Water (17-18 megaOhm) 125 mL |