I was trying to determine the EC50 of Quercetin (range of concentration from 0ug/ml to 300ug/ml) in alpha-glucosidase inhibition assay, by dissolving Quercetin in 99.5% methanol and did the inhibition assay through the following procedure,
A reaction mixture containing 250 μL of 100 mM potassium phosphate buffer (pH 7.0), 150 μL of 0.5 mM 4-nitrophenyl α-D-glucopyranoside, 50 μL of Quercetin, and 150 μL of α-glucosidase (from Saccharomyces cerevisiae; 0.1 unit/mL in 10 mM potassium phosphate buffer, was incubated at 37°C for 30 min. The reaction was ended by adding 600 μL of 200 mM Na2CO3. The absorbance reading was taken at 400 nm. A blank was prepared for each measurement by replacing α-glucosidase with 10 mM potassium phosphate buffer
However, when I was measuring the absorbance at 400nm, the absorbance values were dropping, for both blank and sample (with α-glucosidase), at rough rate of 0.001 per second.
I am seeking for advice here if anything went wrong based on my procedure? or because quercetin was not supposed to dissolve in methanol but some other types of solvent?
Is the colour reducing inside the spectrophotometer, or also on the bench - is this real (can you see less colour after a couple of hours) or is it an artefact of your spectrophotometer? If the latter then use the reading you get as soon as you insert the sample in the spectrophotometer.
Your assay sounds reasonable. However there is quite a high concentration of methanol - you might like to try DMSO instead. If you include standard concentrations of 4-nitophneol, so you can calculate concentration of substrate released, then you should get an idea of any loss of colour during the readings, and any reduction can be calculated for.
1-Deoxynojirimycin is a potent inhibitor of α-glucosidase (Ki ~1 μM) and could be used as a positive control.
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