ATP/NADH-enzyme coupled ATPase assay
Nathalie Jacobs, Peter Vangheluwe, Jialin Chen, Marijke De Jaeger
Abstract
ATP/NADH-enzyme coupled ATPase assay to determine activation of purified ATPase protein via kinetic absorbance measurement.
Steps
Purify the ATPase protein, flash freeze in liquid N2 and store at -80°C until use.
Use a 384-well clear polystyrene microplate: add 25µL of the substrate to be tested, together with 40µL reaction mix, and add 10µL of 63millimolar (mM) ATP at 7.0 per well, with a final volume of 75µL per well.
Prepare serial dilutions of the substrate to be tested, in a final volume of 25µL per well.
If the substrate is dissolved in DMSO, keep the final DMSO concentration in 75µL reaction 0.2%.
Prepare 40µL of reagent mix per well containing: 50millimolar (mM)MOPS-KOH (7.0 ); 100millimolar (mM) KCl; 30millimolar (mM) MgCl2; 2.4 U/µL pyruvate kinase; 2.4 U/µL lactate dehydrogenase; 1.67millimolar (mM) PEP; and 0.6millimolar (mM)NADH, in the presence or absence of 600ng purified ATPase protein. Keep all compounds and the reagent mix at 4°C .
Add 10µL of 63millimolar (mM) ATP at 7.0 per well, and quickly proceed to the acquisition.
Mix the 384-well microplate for 0h 0m 15s prior kinetic measurement in an absorbance plate reader, set at 25°C.
Measure absorbance at 340 nm, at 25°C for0h 30m 0s to 1h 0m 0s. This results in at least 10 data points in the linear phase that can be plotted out over time to determine the OD 340 slope reduction.
