E. coli MurD Assay Kit Plus-100

Mfr.No.	MURD100KE
Description	MurD is a D-Glutamic acid-adding enzyme in the pathway for bacterial cell-wall peptidoglycan synthesis. It is an essential enzyme and attractive target for anti-bacterial drug discovery. MurD catalyses the addition of D-glutamic acid to UDP-MurNAc-L-Ala, generating UDP-MurNAcdipeptide. The ligation reaction uses ATP hydrolysis as an energy source forming ADP and inorganic phosphate. The E. coli MurD Assay is based on measurement of the inorganic phosphate generated from the MurD reaction. The inorganic phosphate is detected by light absorbance at 650 nm. The assay reactions and detection can be performed by using 384-well or 96-well assay plates. Alternatively, the assay reaction can be carried out in Eppendorf tubes and the signal is measured using a cuvette. The high throughput assay can be used for screening inhibitors of E.coli MurD in drug discovery research. It may also be used for characterization of E.coli MurD. The E. coli MurD Assay Kit Plus-100 (Catalog No. MURD100KE) contains the reagents for 100 assays in a 384-well plate assay format including 400 µl of 10 x Buffer, 35 µl of 100 x UDP[1]MurNAc-L-Ala (UMA), 35 µl of 100 x D-Glu, 35 µl of 100 x ATP , 35 µl of 100 x E. coli MurD (2000 nM) and 5 ml of Dye MPA3000 for phosphate detection.
Drug target assays	Antibacterial drug targets

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 261 µl of H2O, 33 µl of 10 x Buffer and 3.3 µl of 100 x E. coli MurD. (2) Prepare 33 µl of 10 x Enzyme substrate by mixing 3.3 µl of 100 x UMA, 3.3 µl of 100 x D-Glu, 3.3 µl of 100 x ATP, and 23.1 µl 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.

Have you used this product?

Combination SKU	Supplier No.	Size	Your price	Quantity
BHE16300050-1	MURD100KE	100 assays	$613.14		Add to Cart
BHE16300050-2	MURD500KE	500 assays	$1957.08		Add to Cart