Protein construct: Wild-type E. coli D-Alanine: D-Alanine Ligase purified from a bacterial expression system.
MW: 39 kDa
Enzyme concentration: 200 nM
Enzyme activity assay: The D-Alanine: D-Alanine Ligase activity is measured by using the E. coli D-Alanine: D-Alanine Ligase Assay Kit (Catalog No. DDA100K).
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 D-Alanine: D-Alanine Ligase Assay – for 100 assays (Catalog No. DDL-100EC) contains 33 µl of 100 x E. coli D-Ala:D-Ala ligase (200 nM). It is for 100 assays.
Assay protocol using the E. coli D-alanine: D-alanine ligase assay kit
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 D[1]Ala:D-Ala ligase.
(2) Prepare 33 µl of 10 x Enzyme substrate by mixing 3.3ml of 100 x Enzyme substrate with 29.7ml 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.
No Description Available.
Reviews of E. coli D-Ala-D-Ala ligase - for 100 assays