• Kinetic analysis of ATP and GTP hydrolysis catalyzed by ATPases and GTPases
• Discovery and characterization of ATPase/GTPase inhibitors
• Measure Pi content of a biological buffer or solution

Kit contents (96 assays)

• ELIPA reaction buffer
• ELIPA reagent 1
• ELIPA reagent 2
• 0.5 mM phosphate standard
• ELIPA reagent 1 resuspension buffer
• ATP
• GTP

Equipment & materials required

• Temperature-regulated spectrophotometer, kinetic mode, absorbance 360 nm. NOTE: The reaction is based upon a shift in absorbance from 330 nm to 360 nm. If using a filter-based instrument, set the absorbance to 370 nm with a bandwidth ≤ ±10 nm.

The ELIPA kit quickly measures ATPase and GTPase activity by detecting phosphate (Pi) release. An absorbance change from 330 to 360 nm shows Pi levels, giving fast, accurate results for a wide range of enzymes.

Detection mechanism

The assay is based upon an absorbance shift (330 - 360 nm) that occurs when 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is catalytically converted to 2-amino-6-mercapto-7-methylpurine in the presence of inorganic phosphate (Pi). The reaction is catalyzed by purine nucleoside phosphorylase (PNP). One molecule of inorganic phosphate will yield one molecule of 2-amino-6-mercapto-7-methyl purine in an essentially irreversible reaction. Thus, the absorbance at 360 nm is directly proportional to the amount of Pi generated in the reaction.

Key characteristics

• Quantitative assay
• Detection method: kinetic absorbance 360 nm
• Assay volume: 300 µl
• Sensitivity: 1 – 50 nmoles Pi
• Timeframe: Approximately 2 hours