E. coli Biotin Protein Ligase Assay Kit Plus

Mfr.No.	BPL100KE, BPL500KE
Description	Biotin protein ligase (BPL or BirA) is responsible for biotinylation of biotin-dependent proteins. It is an essential enzyme and an attractive target for anti-bacterial drug discovery. The E. coli Biotin Protein Ligase Assay is based on measurement of the pyrophosphate generated from the BirA reaction using hydroxylamine in the place of protein as a biotin receptor. The pyrophosphate is converted to phosphate by pyrophosphatase and the 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. Alteatively, 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 BirA in drug discovery research. It may also be used for characterization of E.coli BirA. The E. coli Biotin Protein Ligase Assay Kit Plus-100 (Catalog No. BPL100KE) 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 Biotin, 35 µl of 100 x hydroxylamine, 35 µl of 100 x ATP, 35 µl of 100 x E. coli BirA (5000 nM), 35 µl of 100 x pyrophosphatase (PPase, 10 U/ml) 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, 3.3 µl of 100 x E. coli BirA and 3.3 µl of 100 x PPase. (2) Prepare 33 µl of 10 x Enzyme substrate by mixing 3.3µl of 100 x Biotin, 3.3 µl of 100 x hydroxylamine, 3.5 µl of 100 x ATP with 23.1µl 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.	Specifications	Size	Your price	Quantity
BHE16300041-1	BPL100KE	enzyme included	100 assay	$604.38		Add to Cart
BHE16300041-2	BPL500KE	enzyme included	500 assay	$2025.58		Add to Cart