Hello
I'm working with alpha-glucosidase assays and having problems calculating the ic50 of my samples. At high concentration, they became very yellow and the color interfere with the yellow that is produced by the p-NPG reaction, and even with negative controls it's not possible to obtain accurate results. At more diluted concentrations the results are good and consistent but they don't reach 50% inhibition. The color from the extracts becomes more yellow at the end of the assay, when i add the na2co3 to increase the pH of the reaction mixture. There's any way to overcome this?
My procedure is very common: incubation of the sample/control with the enzyme for 15 minutes; addition of p-NPG to start the reaction and incubation during 30 minutes; addition of Na2CO3 1M to stop the reaction and to produce the nitrophenolate. The amount of product produced is measured at 405 nm.
Thanks and Best Regards
1- I propose to measure the absorbance of only samples (range low to High). this will give you the absorbance of yellow already present in your sample: Ae (sample)
2- later you-measuring the absorbance of your sample with p-NPG (alpha-glucosidase assays). which gives you the yellow level present in the sample in addition to the yellow developped by the reaction: At (Total)
3- does not forget the normal blank: A0 (white)
therefore the true value of the absorbance A = At - (Ae+A0)
1- I propose to measure the absorbance of only samples (range low to High). this will give you the absorbance of yellow already present in your sample: Ae (sample)
2- later you-measuring the absorbance of your sample with p-NPG (alpha-glucosidase assays). which gives you the yellow level present in the sample in addition to the yellow developped by the reaction: At (Total)
3- does not forget the normal blank: A0 (white)
therefore the true value of the absorbance A = At - (Ae+A0)
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