Protein construct: Wild-type E. coli MurC or UDP-N-acetylmuramic acid : L-alanine ligase purified from a bacterial expression system.
MW: 54 kDa
Enzyme concentration: 5 µM MurC with 8 µM UDP-MurNAc for stabilization.
Enzyme activity assay: The MurC activity is measured by using the E. coli MurC Assay Kit Plus-100 (Catalog No. MURC100KE)
Storage temperature: -20 or -80°C. Do not freeze-and-thaw repeatedly.
Enzyme dilution: Use the 1 x assay to dilute the enzyme just before the assay. Do not store diluted enzyme solution
The E. coli MurC – for 100 assays (Catalog No. MURC-100EC) includes 33 µl of 100 x E. coli MurC (5 µM).
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 MurC.
(2) Prepare 33 µl of 10 x Enzyme substrate by mixing 3.3 µl of 100 x UDP-MurNAc, 3.3 µl of 100 x L-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.