ATP synthase Enzyme Activity Microplate Assay Kit (ab109714)

  - ATP synthase Enzyme Activity Microplate Assay Kit (ab109714)

Figure 1. ATP synthase functions by hydrolyzing ATP to ADP and phosphate. This production of ADP is ultimately coupled, as shown in the reaction schematic, to the oxidation of NADH to NAD+ which is monitored as a decrease in absorbance at 340 nm (this assay is fully described in Lotscher, DeJong, and Capaldi (1984) Biochemistry 23, 4134-40). The ATP hydrolysis activity and therefore the coupled reaction is inhibited by oligomycin (a specific inhibitor of ATP Synthase). PK- Pyruvate kinase, LDH-Lactate dehydrogenase, PEP-phosphoenolpyruvate.

  Functional Studies - ATP synthase Enzyme Activity Microplate Assay Kit (ab109714)

Abcam's enzyme activity assays apply a novel approach, whereby target enzymes are first immunocaptured from tissue or cell samples before subsequent functional analysis. All of our ELISA kits utilize highly validated monoclonal antibodies and proprietary buffers, which are able to capture even very large enzyme complexes in their fully-intact, functionally-active states.

Capture antibodies are pre-coated in the wells of premium Nunc MaxiSorp™ modular microplates, which can be broken into 8-well strips. After the target has been immobilized in the well, substrate is added, and enzyme activity is analyzed by measuring the change in absorbance of either the substrate or the product of the reaction (depending upon which enzyme is being analyzed). By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be understood.

  - ATP synthase Enzyme Activity Microplate Assay Kit (ab109714)

Figure 2. The activity of the ATP synthase enzyme is coupled to the molar conversion of NADH to NAD+ measured as a decrease in absorbance at OD 340 nm. The activity rate is expressed as the change in absorbance at 340 nm/minute/amount of sample loaded into the well. To do this, examine the rate of decrease in absorbance at 340 nm over time. This assay starts slowly and takes time to stabilize. The fastest, most linear rate of activity is most often seen between 12 and 30 minutes. As shown, the rate is calculated between these time points. Rate (mOD/min) = (Absorbance 1 - Absorbance 2) / Time in minutes.

  - ATP synthase Enzyme Activity Microplate Assay Kit (ab109714)

Figure 3. For the control or normal sample, the rate versus amount loaded can be plotted as a straight line in the linear region of the assay as shown. Compare the rates of the control (normal) sample and with the rate of the null (background) and with your unknowns, experimental or treated samples to get the relative ATP synthase activity.

  - ATP synthase Enzyme Activity Microplate Assay Kit (ab109714)

Figure 4. Examples of activity/load relationships for cultured cell lysate, rat liver mitochondria, and bovine heart mitochondria.