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NOVOCIB's PRPP Assay Kit is designed to measure PRPP (α-D-5-phosphoribosyl-1-pyrophosphate) content in samples. This enzymatic assay is based on a coupled reaction involving Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and Inosine Monophosphate Dehydrogenase (IMPDH). PRPP is highly unstable, which renders the preparation of accurately weighted standards difficult. NOVOCIB's PRPP Assay Kit has the advantage of measuring PRPP concentration in the sample by absorbance through a stoichiometric NADH2 formation, thus allowing a direct and absolute measurement of PRPP content with no need of any calibration curve. Download our User Manual
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Principle
PRECICE® PRPP Assay Kit is based on the coupled following reactions:
• In the presence of Hypoxanthine, the 5-phosphoribosyl group of PRPP is first transferred by Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) to form Inosine Monophosphate (IMP) and release a pyrophosphate:
• IMP is immediately oxidized to Xanthosine Monophosphate (XMP) by a highly active IMPDH in the presence of NAD.
The NADH2 formed is equivalent to the amount of PRPP in the assay. NADH2 formation is directly monitored spectrophotometrically at 340 nm.
Assay with a range of PRPP standards:
Figure 1 shows the correlation between PRPP concentrations of standards ranging from 2 to 400µM in 200µl-well assays and the PRPP concentrations as measured spectrophotometrically at 340nm by applying a 0.625 cm light path
Kit content:
• Reaction buffer x10
• Reagents: NAD, Hypoxanthine
• IMPDH enzyme, lyophilized (recombinant IMPDH from Staphylococcus aureus, produced in E. coli)
• HGPRT enzyme, lyophilized (Human recombinant HGPRT, produced in E. coli) (^Top)
PRECICE® PRPP Assay Kit is based on the coupled following reactions:
• In the presence of Hypoxanthine, the 5-phosphoribosyl group of PRPP is first transferred by Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) to form Inosine Monophosphate (IMP) and release a pyrophosphate:
• IMP is immediately oxidized to Xanthosine Monophosphate (XMP) by a highly active IMPDH in the presence of NAD.
The NADH2 formed is equivalent to the amount of PRPP in the assay. NADH2 formation is directly monitored spectrophotometrically at 340 nm.
Assay with a range of PRPP standards:
Figure 1 shows the correlation between PRPP concentrations of standards ranging from 2 to 400µM in 200µl-well assays and the PRPP concentrations as measured spectrophotometrically at 340nm by applying a 0.625 cm light path
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Figure 1: Correlation between PRPP concentrations of the standards and PRPP concentrations measured with NOVOCIB's PRPP Assay Kit.
Assays were done in duplicate using a range of freshly prepared PRPP (Sigma-Aldrich, P8296). The concentration of the standards indicated on the graphic takes into account the 75% purity indicated by the the supplier. Due to PRPP instability, standards may be difficult to prepare. This correlation curve shows that a direct measurement of PRPP content can be done without any standard but directly through the absorbance of the NADH2 formed in the assay after 35 minutes of reaction. |
Kit content:
• Reaction buffer x10
• Reagents: NAD, Hypoxanthine
• IMPDH enzyme, lyophilized (recombinant IMPDH from Staphylococcus aureus, produced in E. coli)
• HGPRT enzyme, lyophilized (Human recombinant HGPRT, produced in E. coli) (^Top)
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