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S. aureus PncB Assay Kit Plus

BHE16300140

This product includes the reagents for 100 assays in a 384-well plate assay format including 0.400 ml of 10 x Buffer, 0.035 ml of 100 x NA, 0.035 ml of 100 x PRPP, 0.035 ml of 100 x ATP, 0.035 ml of 100 x S. aureus PncB (5000 nM), 0.035 ml of 100 x pyrophosphatase (PPase, 10 U/ml), and 5 ml of Dye for phosphate detection.

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Product Manual Datasheet
Protocols Description ASSAY PROTOCOL
The following assay protocol is based on the 384-well plate assay format. The reaction volume is 30 µl and the final assay volume is 75 µl. For 96-well plate assays, the reaction volume is 60 µl and the final assay volume is 150 µl. For detection using a cuvette, the reaction volume is 400 µl and the final assay volume is 1000 µl.
 
1. Reagent preparation:
For each 10 assay reactions,
(1) Prepare 297 µl of premix composed of 257.4 µl of H2O, 33 µl of 10 x Buffer, 3.3 µl of 100 x S. aureus PNCB and 3.3 µl of 100 x PPase .
(2) Prepare 33 µl of 10 x Enzyme substrate by mixing 3.3µl of 100 x NA, 3.3 µl of 100 x PRPP, 3.3 µl of 100 x ATP with 23.1µl of water.
 
2. Reaction:
Mix 27 µl of the premix with 3 µl of the 10 x Enzyme substrate in each well. Incubate the reaction mixture at 37°C for 60 min.
 
3. Detection:
Add 45 µl of the Dye MPA3000 into the 30 µl of the reaction mixture. Incubate for 5 min. Measure the light absorbance at 650 nm.
 
ASSAY LINEARITY TEST
Follow the same protocol described above except mixing 27 µl of the premix with 3 µl 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 10 µM.
 
1. In 8 assay wells, add 0.6 µl 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 0.6 µl of water or DMSO. In another control well, add 0.6 µl of 1 M EDTA.
2. Prepare 297 µl of premix and 33 µl of 10 x Enzyme substrate as described above.
3. Mix 26.4 µl of the premix and 0.6 µl of the 50 x inhibitor for 5 min.
4. Add 3 µl of the 10 x Enzyme substrate and incubate the assay reaction for the time in the linear range.
5. Add 45 µl of the Dye MPA3000 into the 30 µl 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
BHE16300140-1 PNCB100KS 100 assays $613.14
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BHE16300140-2 PNCB500KS 500 assays $1957.08
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