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HT Fluorescent Homogeneous PARP Inhibition Assay |
Trevigen's Homogeneous PARP Inhibition Assay is a highly sensitive fluorescent
screening assay for the rapid identification of PARP-1 inhibitors in an in vitro
system. This one hour endpoint assay is performed in two successive steps
requiring only the consecutive addition of reaction components. A PARP reaction
is first performed followed by a detection step. Inhibitors are identified by an
increase in fluorescent signal when PARP mediated NAD+ depletion is inhibited.
The level of NAD+ is coupled to a cycling assay involving alcohol dehydrogenase
and diaphorase. Each time NAD+ cycles through these coupled reactions, a
molecule of highly fluorescent resorufin is generated. Alcohol dehydrogenase
(AD) reduces NAD+ to NADH, while the diaphorase cycles NADH back to NAD+ with
the generation of a highly fluorescent resorufin molecule (from the
non-fluorescent substrate, resazurin). The number of cycles can be controlled by
the time of incubation to adjust assay sensitivity, as needed, and the reaction
is terminated by the addition of a stop solution. In addition, the assay can be
used to determine relative IC50 values for PARP inhibitors and is capable of
detecting as little as 10% inhibition of PARP-1 activity. The Homogeneous PARP
Inhibition Assay is designed for the screening of PARP inhibitors using a NAD
concentration below the Km of PARP. Once potential inhibitors are identified,
results can be confirmed using Trevigen's HT Universal PARP Assay kits (cat#
4676-096-K or 4677-096-K).
Features: |
·
Fluorescent, non-radioactive format
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High throughput 96 test size
·
Detects as little as 10% inhibition of PARP-1 activity
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Applications: |
Identification of PARP-1 inhibitors |
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Components: |
Part # |
Component |
Size |
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4690-096-01 |
10X Cycling Enzymes |
600 µl |
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4690-096-02 |
200 nM NAD |
3.5 ml |
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4690-096-03 |
PARP HSA Enzyme 200 ng/ml |
100 µl |
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4690-096-04 |
Activated DNA, 200 ng/µl |
600 µl |
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4690-096-05 |
Resazurin |
600 µl |
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4690-096-06 |
10X Buffer (1M Tris-HCl, pH 8) |
3.5 ml |
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4690-096-07 |
Stop Solution |
6 ml |
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Storage: |
Components are stored at -20°C and Room Temperature |
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