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HT Fluorescent Homogeneous PARP Inhibition Assay

 

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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

·  High throughput 96 test size

·  Detects as little as 10% inhibition of PARP-1 activity

 

Applications:

Identification of PARP-1 inhibitors

 

Components:

Part #

Component

Size

 

4690-096-01

10X Cycling Enzymes

600 µl

 

4690-096-02

200 nM NAD

3.5 ml

 

4690-096-03

PARP HSA Enzyme 200 ng/ml

100 µl

 

4690-096-04

Activated DNA, 200 ng/µl

600 µl

 

4690-096-05

Resazurin

600 µl

 

4690-096-06

10X Buffer (1M Tris-HCl, pH 8)

3.5 ml

 

4690-096-07

Stop Solution

6 ml

 

Storage:

Components are stored at -20°C and Room Temperature

 

 

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Last modified: 05/29/09