Purine Nucleoside Phosphorylase (PNP, 96 assays) - 1 x 50 U

Applications

• Measurement of free phosphate (Pi) in buffers or serum
• Kinetic assay to follow ATP/GTP hydrolysis in a simple homogenous reaction
• Identification/characterization of GTPase/ATPase enzymes

Material

Purine nucleoside phosphorylase (PNP) protein has been produced in a bacterial expression system.;

• Protein tag: N-terminal His tag for purification
• Molecular weight: approximate mwt ~32 kDa
• Formulation: supplied as a lyophilized powder
• Composition: when resuspended to 0.1U/µl, the buffer composition is: 10 mM PIPES pH 7, 17 mM NaCl, 20 µM EDTA, 1% sucrose, and 0.2% dextran

Purity

Protein purity is assessed by scanning densitometry of Coomassie Blue-stained protein on a 4-20% polyacrylamide gel. Purity was determined to be ≥90% pure.

Biological Activity Assay

The biological activity of ELP03 is determined through the generation of a standard Pi curve. Under the experimental conditions, ELP03 can detect down to 3 nmol of Pi and is linear from 3-50 nmol (see datasheet).
The assay is based upon an absorbance shift (330 nm— 360 nm) that occurs when PNP catalytically converts 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) to 2-amino-6-mercapto-7-methylpurine in the presence of inorganic phosphate (Pi). One molecule of Pi will yield one molecule of 2-amino-6-mercapto-7-methylpurine in an essentially irreversible reaction. Hence, in the presence of an ATPase or GTPase generating phosphate, the absorbance at 360 nm is directly proportional to the amount of Pi generated in an enzyme reaction.