Common Name :
Phosphatase
Product Overview :
Phosphatase Assay Kit provides a simple one-step spectrophotometric method for the quantitation of inorganic phosphate (Pi) in solution, including Pi released from enzymatic reactions. This kit enables continuous assay of reactions that generate Pi such as those catalyzed by ATPases and GTPases.
The release of inorganic phosphate is measured through coupling to PNP-XDH enzymatic system. In the presence of inorganic phosphate, PNP (purine nucleoside phosphorylase) catalyzes inosine phosphorolysis leading to the formation of hypoxanthine and P-ribose. XDH enzyme catalyzes irreversible oxidation of hypoxanthine to uric acid with simultaneous reduction of NAD to NADH2, measurable by absorbance at 340nm. The formation of NADH2 is continuously monitored spectrophotometrically at 340nm. The assay is linear in 5-300μM range.
For maximal accuracy, the assays with cell lysates are run with and without inosine 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.
Description :
A phosphatase is an enzyme that uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol. Because a phosphatase enzyme catalyzes the hydrolysis of its substrate, it is a subcategory of hydrolases. Phosphatase enzymes are essential to a myriad of biological functions, because phosphorylation and dephosphorylation serve diverse roles in cellular regulation and signaling. Whereas phosphatases remove phosphate groups from molecules, kinases catalyze the transfer of phosphate groups to molecules from ATP. Together, kinases and phosphatases effect a form of post-translational modification that is essential to the cell's regulatory network. Phosphatase enzymes are not to be confused with phosphorylase enzymes, which introduce phosphate groups into organic molecules. Due to their ubiquity in cellular regulation, phosphatases are an area of interest for pharmaceutical research.
Storage :
Once dissolved, the reagents provided in the kit are not stable and should be stored on ice and used the day of preparation.
Size :
1 plate (96 assays)
Kit Components :
A standard Phosphatase Assay Kit contains:
one 2mL-tube "Cofactor 1" (DTT);
one 2mL-tube "Cofactor 2" (NAD);
one 15mL-tube with "Substrate" (inosine);
one vial "PNP Enzyme";
one vial "XDH Enzyme";
one tube with 1mL of "20x Reaction buffer";
one transparent 96-well plate.
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