Oct 27, 2022
ATP/NADH-enzyme coupled ATPase assay
- 1KU Leuven
External link: https://doi.org/10.3390/biom13020337
Protocol Citation: Jialin Chen, Marijke De Jaeger, Nathalie Jacobs, Peter Vangheluwe 2022. ATP/NADH-enzyme coupled ATPase assay. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4do2gmk/v1
Manuscript citation:
Houdou M, Jacobs N, Coene J, Azfar M, Vanhoutte R, Haute CVd, Eggermont J, Daniëls V, Verhelst SHL, Vangheluwe P, Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System. Biomolecules 13(2). doi: 10.3390/biom13020337
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 06, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70910
Keywords: ASAPCRN, activation of purified atpase protein, purified atpase protein, coupled atpase, kinetic absorbance measurement, atp, enzyme, assay
Abstract
ATP/NADH-enzyme coupled ATPase assay to determine activation of purified ATPase protein via kinetic absorbance measurement.
Purify the ATPase protein, flash freeze in liquid N2 and store at -80 °C until use.
Use a 384-well clear polystyrene microplate: add 25 µL of the substrate to be tested, together with 40 µL reaction mix, and add 10 µL of 63 millimolar (mM) ATP at 7.0 per well, with a final volume of 75 µL per well.
Prepare serial dilutions of the substrate to be tested, in a final volume of 25 µL per well.
If the substrate is dissolved in DMSO, keep the final DMSO concentration in 75 µL reaction 0.2%.
Prepare 40 µL of reagent mix per well containing: 50 millimolar (mM) MOPS-KOH (7.0 ); 100 millimolar (mM) KCl; 30 millimolar (mM) MgCl2; 2.4 U/µL pyruvate kinase; 2.4 U/µL lactate dehydrogenase; 1.67 millimolar (mM) PEP; and 0.6 millimolar (mM) NADH, in the presence or absence of 600 ng purified ATPase protein. Keep all compounds and the reagent mix at 4 °C .
Add 10 µL of 63 millimolar (mM) ATP at 7.0 per well, and quickly proceed to the acquisition.
Mix the 384-well microplate for 00:00:15 prior kinetic measurement in an absorbance plate reader, set at 25 °C .
15s
Measure absorbance at 340 nm, at 25 °C for00:30:00 to 01:00:00 . 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.
1h 30m