Description
Key facts
Detection method:Colorimetric,
Sample types:Cell Lysate, Cell culture extracts, Cell culture supernatant, Tissue, Tissue Extracts,
Reacts with:Rat, Cow, Human,
Assay type:Enzyme activity,
Assay Platform:Microplate reader
Product details:
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.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions-Multi, Appropriate long-term storage conditions-Multi, Storage information-Please refer to protocols
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
ATP synthase also known as Complex V or F1F0-ATPase catalyzes the synthesis of ATP from ADP and inorganic phosphate. The multisubunit enzyme has a mass of approximately 500 kDa and resides in the inner mitochondrial membrane. It also appears in bacterial plasma membranes and the thylakoid membrane of chloroplasts. ATP synthase operates through a rotary mechanism converting a proton gradient into mechanical energy which then converts into chemical energy in the form of ATP.
Biological function summary
ATP synthase functions as a critical component of cellular energy production. As part of the ATP synthase complex it carries out ATP synthesis essential for numerous biological processes. By utilizing a proton-motive force it enables the regeneration of mitochondrial ATP integral for maintaining cellular energy homeostasis. The enzyme's efficient ATP activity highlights its role in ensuring sustained energy supply within cells.
Pathways
ATP synthase plays an important role in the oxidative phosphorylation pathway. This pathway occurs in mitochondria where the enzyme functions alongside other proteins such as NADH dehydrogenase and cytochrome c oxidase. These proteins forming the electron transport chain contribute to establishing the proton gradient required for ATP synthase activity. Additionally ATP synthase activity is closely linked to the citric acid cycle providing the necessary gradient for ATP production.
ATP synthase relates to conditions such as mitochondrial diseases and neurodegenerative disorders. Dysfunctional mitochondrial ATP synthase can lead to energy deficiency and mitochondrial myopathies. It also has connections to proteins like mitochondrial DNA mutations which impact ATP synthase function and cause cellular energy deficits. Furthermore impaired ATP synthase activity can contribute to conditions like Parkinson's disease where energy metabolism disruptions play a role.
