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Product name
ATP synthase Specific Activity Microplate Assay Kit
See all ATP Synthase products (3) ...
Tests
1 x 96 well plate
Sample type
Cell culture supernatant, Cell culture extracts, Tissue, Tissue Extracts, Cell Lysate
Assay type
Enzyme activity
Cross reactivity
Reacts with
Rat, Cow, Human
Product overview
ab109716 rapid multiplexing microplate kit 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. The ATP synthase enzyme is immunocaptured within the wells of the microplate and the enzyme activity is measured by monitoring the decrease in absorbance at 340 nm. Specifically, 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. Subsequently, in these same wells, the quantity of ATP synthase is determined by adding an ATP synthase specific antibody conjugated with alkaline phosphatase. This phosphatase changes the substrate pNPP from colorless to yellow (OD 405 nm) in a time dependent manner proportional to the amount of protein captured in the wells. Included for optimal performance of the assay are buffer, detergent, antibody, substrate, lipid mix and a 96-well microplate pre-coated with monoclonal antibody.
Notes
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.
Tested applications
Functional Studiesmore details
Properties
Storage instructions
Please see Notes section
| Components | Identifier | 96 tests |
|---|---|---|
| 96-well microplate (12 strips) | 1 unit | |
| AP Buffer | Tube 2 | 1 x 20ml |
| AP Development Reagent | Tube 3 | 1 x 400µl |
| AP label 2500 X | Tube B | 1 x 10µl |
| Buffer | Tube 1 | 1 x 15ml |
| Detector Antibody | Tube A | 1 x 1ml |
| Detergent | 1 x 1ml | |
| Lipid Mix | 1 x 6ml | |
| Reagent Mix | 1 x 20ml |
Research areas
Metabolism >> Pathways and Processes >> Mitochondrial Metabolism >> Mitochondrial markers
Signal Transduction >> Metabolism >> Mitochondrial
Signal Transduction >> Metabolism >> Plasma Membrane >> ATPases
Alternative names
- MS541
- MS543
Applications
Show applications key
Our Abpromise guarantee covers the use of ab109716 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
FuncS
ATP synthase Specific Activity Microplate Assay Kit images:
Functional Studies - ATP synthase Specific Activity Microplate Assay Kit (ab109716)
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 Specific Activity Microplate Assay Kit (ab109716)
Figure 1. 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 Specific Activity Microplate Assay Kit (ab109716)
Figure 2. 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 Specific Activity Microplate Assay Kit (ab109716)
Figure 3. Examples of activity/load relationships for cultured cell lysate, rat liver mitochondria, and bovine heart mitochondria.
- ATP synthase Specific Activity Microplate Assay Kit (ab109716)
Figure 4. The quantity of ATP synthase captured in each well is proportional to the amount of alkaline phosphatase activity within each well. The quantity is the change in absorbance at 405 nm/minute/amount of sample loaded into the well. Examine the linear rate of increase in absorbance at 405 nm with time. As shown, the rate is calculated between these time points. Rate (mOD/min) = (Absorbance 1 - Absorbance 2) / Time in minutes.
- ATP synthase Specific Activity Microplate Assay Kit (ab109716)
Figure 5. 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 Specific Activity Microplate Assay Kit (ab109716)
Figure 6. Examples of quantity/load relationships for cultured liver cell lysate, fibroblast lysate, and rat liver mitochondria.
- ATP synthase Specific Activity Microplate Assay Kit (ab109716)
Figure 7. By measuring both quantity and activity of ATP synthase in a sample, the ratio of the two parameters can be calculated. This is the relative specific activity and should be compared to the normal or control.
Protocols
References for ATP synthase Specific Activity Microplate Assay Kit (ab109716)
This product has been referenced in:
- Kambiz N. Alavian Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase Nature Cell Biology 13:1224-1233 (2011).Read more (PubMed: ) »
- Ito E et al. Potential use of cetrimonium bromide as an apoptosis-promoting anticancer agent for head and neck cancer. Mol Pharmacol 76:969-83 (2009).Read more (PubMed: 19654225) »
See all 3 publications for this product
Publishing research using ab109716? Please let us know so that we can cite the reference in this datasheet
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"