Hi,
I am having some problem with the anti-glucosidase assay of the standard acarbose. I have used reported procedures, but still not getting the expected results. According to the protocol, I have incubated the mixture of 10 ul of sample + 20 ul of enzyme (O.1 U/mL) for 30 min. Then, I added 20 ul of PNPG and incubated the solution for another 30 min. To terminate the reaction, I have added 80 ul of 0.2 M of sodium carbonate and the absorbance was read at 405 nm.
The problem is that the solution takes 24h to change from colorless to yellow and the readings are more or less the same at different concentrations of acarbose.
Can you please advice?
Thanks.
Its necessary to follow protocols or original papers that were successfully used by others.
See attached article
Glucosidase is very pH-sensitive, check if you buffer your samples properly. You don't seem to have any activity after 30 min incubation with your sample, because color development should be very quick, not 24 hours. I am not sure what your assay is, but in a typical assay, PNPG is added directly to the main reaction mix, not after 30 min with your favorite sample. More info on glucosidase can be found here:
http://www.toyobo-global.com/seihin/xr/enzyme/pdf_files/129_132BGH_201.pdf
Dear Salma,
I totally agree with Ebot. In page 129 of the paper sent by Ebot you will find the original procedure and a modified one for your anti-glucosidase assay.
Good luck,
Rafik
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