Product Overview :
PRPP-S Assay Kit is designed for continuous monitoring of PRPP synthesis. The assay is based on coupling of two recombinant enzymes : Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and Inosine Monophosphate Dehydrogenase (IMPDH).
The principle of the assay is based on the coupling of the following enzymatic reactions:
(1) In the presence of ATP and P-ribose, PRPP-Synthetase enzyme catalyzes the formation of PRPP.
(2) In the presence of Hypoxanthine (Hx), PRPP is converted to IMP by Hypoxanthine-guanine phosphoribosyltransferase (HGPRT).
(3) IMP is immediately oxidized by a highly active IMPDH in the presence of NAD with simultaneous formation of NADH2 directly monitored spectrophotometrically at 340nm.
The assay is developed for measuring PRPP-S activity in vitro or in cell lysates. For maximal accuracy, the assays with cell lysates are run with and without P-ribose in parallel. The absorbance rate observed in the absence of inosine is used as blank and is subtracted from the absorbance rate measured in its presence.
Storage :
The kit is shipped at room temperature since dry reagents and lyophilized enzymes are stable at room temperature (up to 2 weeks). However, for long time storage the kit should be frozen upon arrival and stored at -20°C.
Kit Components :
Cysteine (12mg, powder);
NAD (17mg, powder)
ATP (33mg, powder)
P-ribose (10mg, powder)
HPRT and IMPDH enzymes, lyophilized
Reaction buffer (glass vial, 10mL) ;
one transparent 96-well plate
Once dissolved, the reagents can be stored at -20°C for three months.
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