E. coli MurF Assay Kit Plus

Mfr.No.	MURF100KE, MURF500KE
Description	MurF is the enzyme that catalyzes the last step in synthesis of UDP-MurNAc-pentapeptide in the pathway of peptidoglycan biosynthesis in bacteria. It is an essential enzyme and attractive target for anti-bacterial drug discovery. MurF adds a dipeptide D-Ala-D-Ala onto the MurE product UDP-MurNAc-tripeptide. The ligation reaction is coupled to the hydrolysis of ATP forming ADP and inorganic phosphate. The E. coli MurF Assay is based on measurement of the inorganic phosphate generated from the MurF 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 MurF in drug discovery research. It may also be used for characterization of E.coli MurF. The E. coli MurF Assay Kit Plus-100 (Catalog No. MURF100KE) contains the reagents for 100 assays in a 384-well plate assay format including 400 µl of 10 x Buffer, 33 µl of 100 x UDP[1]MurNAc-tripeptide (UMAG-DAP), 33 µl of 100 x D-Ala-D-Ala, 33 µl of 100 x ATP , 33 µl of 100 x E. coli MurF (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 MurF. (2) Prepare 33 µl of 10 x Enzyme substrate by mixing 3.3 µl of 100 x UMAG-DAP, 3.3 µl of 100 x D-Ala[1]D-Ala, 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
BHE16300054-1	MURF100KE	100 assays	$613.14		Add to Cart
BHE16300054-2	MURF500KE	500 assays	$1957.08		Add to Cart