This real-time ELIPA assay tracks kinesin ATPase activity by measuring Pi release (absorbance at 360 nm) as kinesin hydrolyzes ATP—fast, simple, and reliable.

• Kinetic measurement of microtubule-activated kinesin ATPase activity
• Discover kinesin inhibitors
• Determination of kinesin inhibitor IC50s

Kit contents (96 assays)

• ELIPA reaction buffer
• ELIPA reagent 1
• ELIPA reagent 2
• 0.5 mM phosphate standard
• ELIPA reagent 1 resuspension buffer
• Kinesin heavy chain motor domain protein
• Pre-formed microtubules
• Paclitaxel
• DMSO

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 microtubule (MT) activated kinesin ATPase activity by detecting phosphate (Pi) release. An absorbance change from 330 to 360 nm shows Pi levels, giving fast, accurate results.

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 an MT activated kinesin reaction.

Key characteristics

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