We are currently working on in vitro glucosidase inhibition potentials of some compounds. The enzyme substrates in our protocol is pNPG. It is difficult to obtain this substrate at this moment. Efficient alternative protocols/substrates are welcome
Sigma also sells a fluorogenic substrate, 4-methylumbelliferyl α-D-glucopyranoside.
You can use a more natural substrate such as amylopectin or maltitol. The enzyme releases glucose which you can then assay using a reducing end assay, such as 2,4-dinitrosalicyclic acid (DNS). Alternatively you can use a glucose specific assay, using glucose oxidase (producing H2O2) or glucokinase/glucose-6-phosphate dehydrogenase (converting NADP to NADPH). These last two are compatible with maltose hydrolysis.
pNP-Glc is very cheap and reliable. Try alternative commercial sources, like Carbosynth or Sigma. Otherwise follow Ellis' advices but it is much work...
hi! does any one have a protocol to assay glucosidase activity with pNP-Glc as a substrate in ectopic proteins immunoprecipited from mamalian cells??
Does anybody know how to use 4-methylumbelliferyl α-D-glucopyranoside for alpha-glucosidase inhibition assay and what is the protocol for the same
hello
Any ideas why when I add the enzyme alpha glucosidase to the
4-NITROPHENYL substrate no staining occurs ?? knowing that the enzyme concentration is 1 unit and the substract 5 mM
Make sure you are using the correct substrate, 4-nitrophenyl-alpha-D-glucoside, not 4-nitrophenyl-beta-D-glucoside.
Adam B Shapiro your point is relevant, you're right, thank you very much,
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