Product Overview :
ITPase Assay Kit is the first non-radioactive and continuous kit designed to measure ITP pryophosphohydrolase content in samples. This enzymatic assay is based on a reaction involving Inosine Monophosphate Dehydrogenase (IMPDH).
The principle of the assay is based on the coupling of the following enzymatic reactions:
(1) In the presence of ITP, ITP pyrophosphohydrolase enzyme catalyzes the formation of IMP.
(2) In the presence of NAD, IMP is immediately oxidized by a highly active IMPDH in the presence of NAD with simultaneous formation of NADH2 directly monitored spectrophotometrically at 340 nm.
The assay is developed for measuring ITPase activity in vitro or in cell lysates.
For maximal accuracy, the assays with cell lysates are run with and without ITP in parallel. The absorbance rate observed in the absence of ITP is used as blank and is subtracted from the absorbance rate measured in its presence.
Description :
ITP pyrophosphatase, or ITPase (EC 3.6.1.19), is an intracellular enzyme that catalyzes the pyrophosphohydrolysis of ITP/dITP and xanthosine triphosphate to prevent unusual nucleoside triphosphates from accumulating in (deoxy) nucleoside triphosphate (d)NTP pools and being integrated into RNA and DNA. This enzyme is encoded by the ITPA gene in mammals. Certain ITPA variants causing ITPase deficiency have been linked to adverse reactions to the immunosuppressive thiopurine drugs azathioprine and 6- mercaptopurine, and protection against ribavirin-induced anemia.
Storage :
Once dissolved, the reagents provided in the kit are not stable and should be stored on ice and used the day of preparation.
Kit Components :
The kit allows to perform 24 analyses in a time (8 samples in triplicate, 12 samples in duplicate).
A standard ITPase Assay Kit contains:
one tube "Cofactor 1";
one tube "Cofactor 2 ";
one tube "Enzymatic mix";
one tube "10X buffer" (pre-filled with 1 ml of 10X buffer);
one 15mL tube "Blank" orange cap;
one 15mL tube "Reaction mixture with ITP" blue cap (pre-filled with 5µmol ITP);
one transparent 96-well plate.
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