Assay Method Information

Assay Name:  Urease Inhibition Assay
Description:  Briefly, 50 mL broth cultures (2.0 × 10^8 CFU/mL) were centrifuged (5000g, 4°C)to collect the bacteria, and after washing twice with phosphate-buffered saline (pH 7.4), the H. pylori precipitation was stored at −80°C. H. pylori was returnedto room temperature, and after the addition of 3 mL of distilled water and protease inhibitors, sonication was performed for 60 s. Following centrifugation(15,000g, 4°C), the supernatant was desalted through SephadexG-25 column (PD-10 columns, Amersham-Pharmacia Biotech, Uppsala, Sweden). The resultant crude urease solution was added to an equal volume of glycerol and stored at 4°C until use in the experiment. The mixture, containing 25 μL (4U) of H. pylori urease (100 mM HEPES, pH 6.8) and 25 μL of the test compounds of various concentrations, was preincubated for 3 h at room temperature in a 96-well assay plate. After preincubation, 0.2 mL of 100 mM HEPES (pH 6.8) buffer containing 500 mM urea and 0.002% phenol red were added and incubated at 37°C.
Affinity data for this assay
 

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