Apr 15, 2026

Measurement of ATP13A4 ATPase activity using ADP-Glo Max Assay

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvo9kxdv4o/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvo9kxdv4o/v1
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Measurement of ATP13A4 ATPase activity using ADP-Glo Max Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9kxdv4o/v1
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: December 09, 2024
Last Modified: April 15, 2026
Protocol  Integer ID: 114640
Keywords: ASAPCRN, ATP13A4, ATPase assay, measurement of atp13a4 atpase activity, atp13a4 atpase activity, atpase activity of atp13a4, atpase activity, atp13a4, atp hydrolysis, glo max assay this protocol, rate of atp hydrolysis, glo max assay, assay, using adp, based adp, promega, luminescence
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol describes a luminescence-based ADP-Glo Max assay (Promega) to measure the ATPase activity of ATP13A4 by detecting the rate of ATP hydrolysis. 
Materials
  • ADP-Glo Max Assay (Cat# V7002; Promega)
  • Purified ATP13A4 (100 ng per reaction)
  • Putrescine (Cat# P7505; Sigma-Aldrich)
  • Spermidine (Cat# S2626; Sigma-Aldrich)
  • Spermine (Cat# 85590; Sigma-Aldrich)
  • MgCl₂ hexahydrate (Cat# M2670; Sigma-Aldrich)
  • KCl (Cat# P9541; Sigma-Aldrich)
  • MOPS (Cat# A1076.1000; VWR)
  • KOH (Cat# 221473; Sigma-Aldrich)
  • Dithiothreitol (DTT; Cat# A2948.0025; VWR)
  • n-Dodecyl-β-D-maltoside (DDM; Cat# 1758-1350; Inalco)
  • Synergy H1 microplate reader (Biotek)
  • GraphPad Prism Software
Before start
Allow ADP-Glo Max Assay components to reach room temperature before use!
Prepare a 25 µl reaction mixture containing: 100 ng purified ATP13A4, 11 mM MgCl2, 100 mM KCl, 50 mM MOPS-KOH (pH 7), 1 mM DTT, 0,001% (wt/vol) DDM, and desired polyamine concentrations.
Incubate the mixture 37 °C  for 00:05:00 .

5m
Add ATP to a final concentration of 5 millimolar (mM) to initiate the reaction.

Incubate the mixture for 01:00:00 at 37 °C .

1h
Following this, incubate the reaction for 01:00:00 at Room temperature .

1h
Add 25 µL of ADP-Glo reagent to each reaction and incubate for 01:00:00 atRoom temperature .

1h
Add 50 µL of ADP-Glo Max Detection Reagent to each reaction and incubate for 01:00:00 in the dark at Room temperature .
Note
Protect samples from light during incubation with ADP-Glo Max Detection Reagent.


1h
Measure luminescence using a compatible microplate reader (e.g. Synergy H1 microplate reader; Biotek).
Analyze the luminescence data using GraphPad Prism Software. Perform curve fitting to evaluate ATPase activity.
Protocol references
van Veen S, Martin S, Van den Haute C, Benoy V, Lyons J, Vanhoutte R, et al. ATP13A2 deficiency disrupts lysosomal polyamine export. Nature. 2020;578(7795):419-24.