Jul 09, 2024
ATPase assay/ADP-glo kit
- 1KU Leuven, Aligning Science Across Parkinson's (ASAP)
Protocol Citation: Rania Abou El Asrar, Peter Vangheluwe 2024. ATPase assay/ADP-glo kit. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4q1x8vo5/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 06, 2023
Last Modified: July 09, 2024
Protocol Integer ID: 91889
Funders Acknowledgement:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000458
Abstract
This protocol is successfully used to assess activity of P5B-ATPases with polyamines as substrate.
Materials
ADP-glo max assay (Promega, V7002)
DDM (Inalco, 1758-1350)
Prepare master mix on ice: 5 µg microsomes, 2.5 µg DDM (Inalco, 1758-1350), reaction buffer (50 mM MOPS-KOH, pH 7, 100 mM KCl, 11 mM MgCl2 supplemented with 1 mM DTT and protease inhibitor (Sigmafast, Sigma, S8830-20TAB).
Incubate master mix for 30 minutes at room temperature in overhead shaker.
Pipet master mix (18.75 µl) into a white 96-well plate.
Prepare serial dilution of the substrate to be tested in 0.1 M MOPS-KOH, pH 7.
Pipet substrate dilution series (5 µl) into the 96-well plate.
Incubate for 5 minutes at room temperature then 5 minutes at 37 °C.
Add 5 mM pure ATP (1.25 µl) to initiate the reaction.
Incubate for 20 minutes at 37 °C followed by 10 minutes at room temperature.
Stop the reaction by addition of 25 µl ADP-glo reagent.
Incubate at room temperature for 40 minutes.
Add 50 µl/well of ADP-glo max reagent.
Incubate in the dark at room temperature for 60 minutes.
Measure luminescence (integration: 500 ms)