Oct 27, 2022

Public workspaceATP/NADH-enzyme coupled ATPase assay

DOI
dx.doi.org/10.17504/protocols.io.6qpvr4do2gmk/v1
  • 1KU Leuven
Nathalie Jacobs
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr4do2gmk/v1
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
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 Temperature-80 °C until use.

Use a 384-well clear polystyrene microplate: add Amount25 µL of the substrate to be tested, together with Amount40 µL reaction mix, and add Amount10 µL of Concentration63 millimolar (mM) ATP at Ph7.0 per well, with a final volume of Amount75 µL per well.

Prepare serial dilutions of the substrate to be tested, in a final volume of Amount25 µL per well.
If the substrate is dissolved in DMSO, keep the final DMSO concentration in Amount75 µL reaction 0.2%.

Prepare Amount40 µL of reagent mix per well containing: Concentration50 millimolar (mM) MOPS-KOH (Ph7.0 ); Concentration100 millimolar (mM) KCl; Concentration30 millimolar (mM) MgCl2; 2.4 U/µL pyruvate kinase; 2.4 U/µL lactate dehydrogenase; Concentration1.67 millimolar (mM) PEP; and Concentration0.6 millimolar (mM) NADH, in the presence or absence of Amount600 ng purified ATPase protein. Keep all compounds and the reagent mix at Temperature4 °C .
Add Amount10 µL of Concentration63 millimolar (mM) ATP at Ph7.0 per well, and quickly proceed to the acquisition.
Mix the 384-well microplate for Duration00:00:15 prior kinetic measurement in an absorbance plate reader, set at Temperature25 °C .

15s
Measure absorbance at 340 nm, at Temperature25 °C forDuration00:30:00 to Duration01:00:00 . 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