ATPase assay/ADP-glo kit
Peter Vangheluwe, Rania Abou El Asrar
Abstract
This protocol is successfully used to assess activity of P5B-ATPases with polyamines as substrate.
Steps
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 MgCl2supplemented 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)
