Assay Report Card

ID: ALA3706365

Type: Binding

Description: Inhibition Assay: To assay the activity of Pseudomonas aeruginosa LpxC enzyme, LpxC was reacted with its substrate UDP-3-O-(R-3-hydroxydecanoyl)-N-acetylglucosamine and the amount of the reaction product was determined by quantifying the amino groups present in it.Specifically, 12.5 ng of Pseudomonas aeruginosa LpxC enzyme (as acquired by preparing chromosomal DNA from Pseudomonas aeruginosa, subjecting the DNA to PCR (polymerase chain reaction) using LpxC specific primers to acquire Pseudomonas aeruginosa LpxC genes, incorporating the genes into a vector, and expressing the same with E. coli) was mixed with 80 .mu.mol/L of UDP-3-O-(R-3-hydroxydecanoyl)-N-acetylglucosamine (Wako Pure Chemical Industries, Ltd.) and the mixture was incubated at room temperature for 40 minutes. The reaction was performed in 40 mmol/L of Hepes buffer solution (pH 8.0) supplemented with 0.02% Bridge 35 and 80 .mu.mol/L of dithiothreitol. To terminate the reaction, 0.2 mol/L of borax was added to the reaction mixture.