ATP synthase Relative Activity Microplate Assay Kit

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ATP Synthase Activity Assay Kit ab109716 is used to determine both the activity and quantity of ATP synthase (Complex V) in a human or rat sample. The ratio of the two measurements represents the enzyme's specific activity.

In the ATP synthase assay protocol, the ATP synthase enzyme is immunocaptured within the wells of a microplate. The conversion of ATP to ADP by ATP synthase is coupled to the oxidative reaction of NADH to NAD+. The formation of NAD+ results in a decrease in absorbance at 340 nm which is proportional to the amount of enzyme activity.

Subsequently, the quantity of ATP synthase is determined by adding an anti-ATP synthase antibody conjugated with alkaline phosphatase. Alkaline phosphatase acts on the substrate pNPP to form a yellow color (OD 405 nm) proportional to the amount of ATP synthase captured in the wells.

ATP synthase assay protocol summary:
- add samples to wells and incubate for 3 hrs to capture enzyme
- wash
- add lipid mix and incubate for 45 min
- add reaction mix
- analyze enzyme activity with microplate reader for 1-2 hrs at 1 min intervals
- empty wells and incubate with solution A for 1 hr
- wash
- add solution B and incubate for 1 hr
- wash
- add development solution
- analyze enzyme quantity with microplate reader for 30 min at 1 min intervals

Store Buffer, Detergent, Detector Antibody, AP Label, AP Buffer, and the microplate at 4°C. Store the Reagent Mix, AP Development Reagent and Lipid Mix at -20°C. For multiple experiments, the Reagent Mix and AP Development Reagent may be aliquoted and stored at -20°C, or at -80°C for long term storage.