Hello everyone....

I am working on urease inhibition, and I have used indophenol method for screening my compounds.

Thiourea is a positive control and standard inhibitor in my experiment.

Although IC50 of Thiourea is about 21 in articles, my positive control even in high concentrations like 1mM turns blue.

I've tried different brands of Thiourea, and the results were the same.

I have carried out the experiment over and over based on the protocols that are written in the articles.

and one of the protocols is:

The assay mixture included urea (850 μL, 30mM, in phosphate

buffer 100 mM, pH 7.4), compounds 7a-l (100 μL, at the concentration of 0–10 mg/ml), and phosphate buffer(100 mM, pH 7.4) with the total volume of 985 μL. Then, 15 μL of urease solution (JBU, EC 3.5.1.5, 3 mg/mL, the phosphate buffer 100 mM, pH 7.4) was added to the assay mixture, and the obtained mixture was incubated at 37 °C for

60 min. After that, the concentration of ammonia produced by the activity of uninhibited urease enzyme was determined through addition of solution A (500 μL, consist of 1% w/v

phenol (5.0 g) and 0.005% w/v sodium nitroprusside (25.0 mg) in 500 mL distilled water) and solution B (500 μL,consist of 0.5% w/v sodium hydroxide (2.5 g) and 4.2 mL

sodium hypochlorite (5% chlorine) in 500 mL distilled water)and further incubated at 37 °C for 30 min.

I would appreciate your help.