Jul 20, 2022
ADP Glo Max measuring ATP13A2 ATPase activity in microsomes
- Sue Sim1,
- eunyong_park 1
- 1University of California, Berkeley
Protocol Citation: Sue Sim, eunyong_park 2022. ADP Glo Max measuring ATP13A2 ATPase activity in microsomes. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr65ozvmk/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: July 17, 2022
Last Modified: July 20, 2022
Protocol Integer ID: 66882
Keywords: atp13a2 atpase activity in microsome, adp detection assay, based adp detection assay, measuring atp13a2 atpase activity, microsome, commercial luminescence
Abstract
Measuring ATP13A2 ATPase activity in microsomes using commercial luminescence-based ADP detection assay (ADP Glo Max; Promega)
Materials
Luminescence-based ADP detection assay (ADP Glo Max assay; Promega)
Reaction Buffer
50 mM MOPS-KOH pH 7.0
100 mM KCl
11 mM MgCl2
1 mM DTT
0.02% DDM
0.001-10 mM spermine
Troubleshooting
Thaw microsomes on ice
Use 1 μg microsomes per reaction (1 µL of 5 mg/mL stock)
Dilute to 4 µL in Reaction Buffer at 4C
Equilibrate microsomes in reaction buffer for 01:30:00 On ice and for 00:10:00 at 37 °C
1h 40m
Add 1 µL of 25 mM ATP (final concentration 5 mM ATP) to start reaction (total reaction volume 5 µL)
Incubate for 00:15:00 at 37 °C upon addition of ATP
15m
Terminate reaction by heating samples for00:05:00 at 80 °C
5m
Mix with 5 µL of ADP-Glo Reagent for 60-80 min at23 °C
Mix with 10 uL of ADP-Glo Max Detection Reagent for 01:00:00
1h
Measure luminescence in 384 multi-well plate using a luminometer (NOVOstar; BMG Labtech)