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PRECICE® HPRT Assay kit is a simple non-radioactive assay for measurement of Hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in a convenient 96-well plate format. In the assay, HPRT activity is measured as a rate of production of IMP, which is oxidized by recombinant IMP dehydrogenase with concomitant formation of NADH2. The formation of NADH2 is continuously monitored spectrophotometrically at 340nm. The assay is linear up to 340nmol/hour/ml of HPRT activity, enables the detection of al low as 6.75nmol/hour/ml and provides results with uncertainties of 6.5%. Specific activities of HPRT in lysates of erythrocytes and cultured cells measured by PRECICE® HPRT Assay kit are comparable to those obtained previously by radiochemical procedure.  Find the Application Note advertized by Nature Methods
or download it in a PDF format. Download our User Manual
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Principle
IMP dehydrogenase enzyme catalyzes the irreversible oxidation of IMP to XMP with simultaneous reduction of NAD to NADH2. PRECICE® HPRT Assay Kit takes advantage of this property for monitoring the activity of HPRT. In the assay, HPRT activity is measured as a rate of production of IMP, which is immediately oxidized to XMP by IMPDH with concomitant reduction of NAD to NADH2 measurable by absorbance at 340nm.
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Accurate estimation of HPRT activity with NOVOCIB's IMPDH-based assay is done:
• in unidirectional reaction where IMP produced is immediately converted by IMPDH to XMP;
• through continuous monitoring of HPRT activity at 340nm in a convenient 96-well plate;
• by simultaneous analysis of 15 samples in triplicate per plate (2h);
• without sample preparation or prior inactivation of cellular 5’-nucleotidase to prevent IMP dephosphorylation.
The assay is simple to use (“add-and-measure”), rapid (2h), reliable (total imprecision 6.5%) and cost-effective.
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Calibration curve of IMPDH-based PRECICE® HPRT Assay
The rate of the increase in absorbance at 340nm per hour as a function of the concentration of human recombinant HPRT enzyme (NovoCIB, ref. E-Nov-9). The changes in absorbance corresponding to 3 different control hemolysates diluted in complete reaction mixture to final hemoglobin concentration 1mg/ml (n=4) are shown by filled squares, filled triangles and filled circles. The insert shows a linear correlation observed in whole range of 21ng/ml up to 1.5µg/ml of recombinant HPRT; the units are the same. |
| Kit Content: | • Bacterial IMPDH
• IMPDH Reconstitution Buffer |
• NAD (100x)
• HKM Buffer containing hypoxanthine(10x) |
| Optional: • Human recombinant HPRT enzyme for preparing standard solution. | ||
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