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E. coli NAD+ Kinase Assay Kit Plus

BHE16300133

This product includes 600 ul of 10 x assay buffer, 35 ul of 100 x NAD+, 35 ul of 100 x E. coli NAD kinase, 35 ul of 100 x ATP, 35 ul of 100 x MUK Reagent A, 35 ul of 100 x MUK Reagent B and 300 ul of 10 x fluorescence dye for 100 assays in a 384-well assay format.

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Product Manual Datasheet
Protocols Description Assay Protocol
1. Reagent preparation:
(1) 1 x reaction buffer: dilute the 10 x Reaction Buffer 10-fold with water. The 1 x reaction buffer is composed of 50 mM Tris-HCl, pH 8.0, 3 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 0.003% Brij-35.
(2) 10 x NAD+ : dilute the 100 x NAD+ solutions (100 mM) 10-fold with water.
(3) 10 x ATP: dilute the 100 x ATP solutions (2 mM) 10-fold with water.
(4) 10 x NAD+ kinase: dilute the 100 x NAD+ kinase 10-fold with 1 x reaction buffer.
(5) 10 x MUK Reagent A: dilute the 100 x MUK Reagent A 10-fold with water.
(6) 10 x MUK Reagent B: dilute the 100 x MUK Reagent B 10-fold with 1 x reaction buffer.
(7) 1 x fluorescence dye: dilute the 10 x Fluorescence dye 10-fold with water.
 
2. Kinase assay
The kinase reaction volume is 30 µl.
(1) In a standard black 384-well plate (Matrix 4318), mix 12 µl of water, 3 µl of 10 x reaction buffer, 3 µl of 10 x NAD+ , 3 µl of 10 x ATP, 3 µl of 10 x kinase. Incubate the reaction for 2 min.
(2) Add 3 µl of 10 x MUK Reagent A, 3 µl of 10 x MUK Reagent B.
(3) Incubate the reaction mixture at 37°C for 60 min.
(4) Add 30 µl of the 1 x fluorescence dye into the 30 µl of the reaction mixture.
(5) Measure the fluorescence intensity at 535 nm with excitation at 485 nm.
 
Assay Protocol for enzyme inhibition
In order to accurately measure the IC50, it is important to make sure the assay is in the linear range. The linear range can be defined by enzyme concentration-dependence and time-dependence experiments. The enzyme concentration and time in the linear range is selected for the IC50 measurement.
 
For the enzyme inhibition experiment, typically 50 x stock solutions with a 2-fold serial dilution in water or DMSO are prepared. In the wells of the 384-well plate, 0.6 µl of the 50 x inhibitor is mixed with a premix composed of the buffer and enzyme for 5 min. Then the substrate is added. After incubation for 2 min, 3 µl of 10 x MUK Reagent A and 3 µl of 10 x MUK Reagent B are added. The total assay volume is 30 µl. At the end of the reaction, the dye is added and the fluorescence intensity is measured.
Combination SKU Supplier No. Size Your price Quantity
BHE16300133-1 NADK100KE 100 assays $556.13
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BHE16300133-2 NADK500KE 500 assays $1863.89
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