PURETIME ® caspase assays
Key Features Key Benefits
- Enzyme and substrate additions only - One step
- Fluorescent assay - 384 well plate volume or smaller
- Customised peptide sequences - Target enzyme of interest
- Fluorescence intensity or lifetime discriminated - Resists interference
- Instant measurement of caspase activity - Kinetic measurements easily made
Assay principle
Fluorescence of PURETIME®17 is enhanced upon cleavage, producing more intense emission and a shorter fluorescence lifetime. The small fluorophor does not adversely affect the action of the enzyme. Fluorescence measurements can be made at any point during the caspase incubation, permitting time course experiments and direct determination of reaction kinetics.
Examples
Caspase 3 activity assay
Caspase 3 enzyme titration using SDEVD-PURETIME17 caspase substrate (1000nM)
(Lifetime discriminated intensity mode)
Caspase 3 enzyme activity was readily quantified by measuring the fluorescence intensity of the shorter lifetime PURETIME17 cleavage product (plotted above as Int2 (6.3ns)). A good linear range and sensitivity was demonstrated.
Caspase 3 kinetic assay
Caspase 3 time course using SDEVD-PURETIME17 substrate (1000nM)
(Lifetime discriminated intensity mode)
Curve-fitting to a first order rate equation gave a rate constant of 9.3 counts per minute at t=zero and a limiting signal of 671 counts. Knowing the number of mols of substrate in the 100micL well volume, the initial reaction rate of the caspase 3 enzyme was found to be 9.5micM per minute per mg, in good agreement with the enzyme certificate of analysis.
Caspase 3 inhibition measurement
Dose response curves for specific caspase 3 inhibitor I3 and
universal caspase inhibitor IU with SDEVD-PURETIME17 caspase substrate (1000nM)
Dose response curves were recorded over six decades of inhibitor concentration. The caspase 3 specific inhibitor I3 (Z-DEVD-FMK) was found to have an IC50 = 110nM whereas the universal caspase inhibitor IU (Boc-D-FMK) had an IC50 = 1200nM.
PURETIME17 caspase 3 assay technology has been shown to be well-suited to quantitative measurement of caspase activity, caspase kinetics, rank ordering of inhibitors and quantification of inhibition.
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Products
| Product code | N terminus | Sequence | C terminus | Amount |
| PT17-CA1-P | Ac- | YVAD | -
PURETIME
® 17 |
1mg |
| PT17-CA3-P | Ac- | SDEVD | -
PURETIME
® 17 |
1mg |
| PT17-CA8-P | Ac- | IETD | -
PURETIME
® 17 |
1mg |
| PT17-CA9-P | Ac- | LEHD | -
PURETIME
® 17 |
1mg |
Ordering
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