ADP Glo Max measuring ATP13A2 ATPase activity in microsomes

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 1h 30m 0s On ice and for 0h 10m 0s at 37°C

Add 1 µL of 25 mM ATP (final concentration 5 mM ATP) to start reaction (total reaction volume 5 µL)

Incubate for 0h 15m 0s at 37°C upon addition of ATP

Terminate reaction by heating samples for0h 5m 0s at 80°C

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 1h 0m 0s

Measure luminescence in 384 multi-well plate using a luminometer (NOVOstar; BMG Labtech)