Dear all,
I tried to evaluate an alpha glucosidase assay of the methanol plant extract using this protocol: 50µL of sample (100-1000 µg/ml) + 100 µL of α-glucosidase (0.2 u/ml) + 50 µL of p-NPG (2.5 mM), incubated for 20 min at 37 °C then the reaction quenched by addition 1 ml of Na2CO3 (0.1 M), the absorbance recorded at 405 nm.
The control with buffer obtained a yellow colour with Abs= 0.6, but when I tried to use my sample the colour was appeared even I use different sample concentrations. I dissolved the samples in DMOS and dilutions have be done unsing buffer. In case that sample diluted using MeOH the colour of the same sample concentrations disappeared.I tried to change the concentration of enzyme and NPG but I got the same problem. My samples have yellow colour in the highest concentrations so I tried to work with the diluted samples.
Is anyone have explanation and solution of this problem.
Thanks Mohamed Cheurfa, but my problem is my samples have a yellow colour and the reaction should give also yellow colour. Howover, when I tried to decrease the concentration to get a colorless solution from my samples, I have yellow colour from the reaction (that is mean less 50% inhibition) but I can not reach to colorless (more than 50% inhibition). I did the sample dilution in buffer is it OK or should I dilute them in DMSO or MeOH?
Hi mahmoud
you can try dilution in MeOH or DMSO and compare the results
good luck
When I did dilution with MeOH, I didn't get a yellow colour for the same sample concentrations that I used to dilute the sample with buffer, is this normal?
Because I tried to do the control using MeOH instead of buffer but I did not get a yellow colour
Dear mahmoud
I give you a contact of a researcher who can help you better
Dr: Sung-Yum Seo :[email protected]
best regards
Thank you Mohammed for your interest and help. OK I will contact him.
see below method
http://www.thenanoresearch.com/upload/justPDF/0573.pdf
You may like to check out the following sources:
Andreae-Marobela K, Ntumy AN, Makobela M, Dube M, Sosome A, et al. (2012) "Now I heal with pride" - The application of screens-to-nature technology to indigenous knowledge systems research in Botswana: implications for drug discovery. In Drug discovery in Africa: impacts of genomics, natural products, traditional medicines, insights into medicinal chemistry, and technology platforms in pursuit of new drugs. Edited by Chibale K, Davies-Coleman M, Masimirembwa C. Heidelberg: Springer-Verlag: 239-264.
Harisha H (2006) An intrduction to practical biotechnology. Laxmi Publications. New Delhi 3:544.
Thanks
Hello
I'm working with alpha-glucosidase and having the same problem. My samples became yellow at the higher concentrations, and i only get results at the lower concentrations. But i need to calculate de IC50 and the diluted samples don't reach 50% inhibition. How did you overcome this problem? My procedure is very similar to yours.
Thanks and Best Regards
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