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H. influenzae DNA Helicase ATPase assay Kit Plus-100

BHE16300065

This product includes 0.500 ml of 10 x assay buffer, 0.035 ml of 100 x DNA, 0.030 ml of 100 x H. influenzae DNA helicase, 0.035 ml of 100 x ATP and 5 ml of dye for 100 assays in a 384-well assay format.

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Product Manual Datasheet
Protocols Description Assay Protocol
1.Reagent preparation:
For each 10 assay reactions,
(1) Prepare 297µl of premix composed of 261µl of H2O, 33µl of 10 x Buffer and 3.3µl of 100 x H. influenzae DNA helicase.
(2) Prepare 33µl of 10 x Enzyme substrate by mixing 3.3µl of 100 x ATP and 3.3µl of 100 x DNA and 26.4µl of water.
 
2.Reaction:
Mix 27µl of the premix with 3µl of the 10 x Enzyme substrate in each well. Incubate the reaction mixture at 37°C for 60 min.
 
3.Detection:
Add 45µl of the Dye MPA3000 into the 30µl of the reaction mixture. Incubate for 5 min. Measure the light absorbance at 650 nm.
 
Note: The final concentrations for the ATPase assays of the helicases are 20 mM HEPES, pH 7.5, 20 mM potassium glutamate, 1 mM DTT, 0.005% Triton X-100, 10 mM MgCl2, 20µg /ml DNA, 0.25 mM ATP and 200 nM DNA helicase. A negative control reaction can be the reaction mixture without addition of ATP or enzyme.
 
ASSAY LINEARITY TEST
Follow the same protocol described above except mixing 27µl of the premix with 3µl of the 10 x Enzyme substrate at different time points. Plot the reaction signal versus the reaction time to define the linear range.
 
IC50 MEASUREMENT OF ENZYME INHIBITORS
The concentration range of the inhibitor to be tested depends on the potency of the inhibitor. In general, the maximum concentration is about 10 to 20 fold higher than the IC50 value. The following protocol is for IC50 measurement of one inhibitor with IC50 values around 10µM.
 
1. In 8 assay wells, add 0.6µl of 2-fold serial dilution solutions of the inhibitor from 5 mM to 0.039 mM in water or DMSO. In one control well, add 0.6µl of water or DMSO. In another control well, add 0.6µl of 1 M EDTA.
2. Prepare 297µl of premix and 33µl of 10 x Enzyme substrate as described above.
3. Mix 26.4µl of the premix and 0.6µl of the 50 x inhibitor for 5 min.
4. Add 3µl of the 10 x Enzyme substrate and incubate the assay reaction for the time in the linear range.
5. Add 45µl of the Dye MPA3000 into the 30µl of the reaction mixture. Incubate for 5 min. Measure the light absorbance at 650 nm.
6. Calculate IC50s using a computer IC50 fitting software.
Combination SKU Supplier No. Size Your price Quantity
BHE16300065-1 DNAB100KH 100 assays $613.14
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BHE16300065-2 DNAB500KH 500 assays $1957.08
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