Invitrogen™ ATP Determination Kit 

Description

The ATP Determination Kit is a highly sensitive luminescence assay designed for the quantitative detection of ATP levels in biochemical and cell viability assays. This ATP assay kit utilizes the reaction between recombinant firefly luciferase and its substrate D-luciferin and is based on luciferase’s absolute requirement for ATP in producing light (emission maximum approximately 560 nm at pH 7.8). With its exceptional sensitivity, capable of detecting as little as 0.1 picomole of ATP, this assay is well-suited for kinetic studies and can be used with luminometers or microplate readers equipped with a luminescence mode, such as the Thermo Scientific Varioskan ALF and LUX Multimode Microplate Readers.

This luciferase ATP assay includes sufficient reagents for ten 96-well plates and offers a unique advantage over other commercially available ATP detection kits by providing the luciferase and luciferin components separately, allowing researchers to optimize the reaction conditions for their specific instruments and samples.

Features of the ATP Determination Kit include:

• Utilizes a luminescence-based system to monitor light emission, enabling sensitive and accurate quantification of ATP levels
• Offers exceptional sensitivity, capable of detecting as little as 0.1 picomole of pre-existing ATP or ATP generated in kinetic systems
• Employs a unique formulation where the luciferase and luciferin components are packaged separately, allowing researchers to optimize assay conditions for their specific samples and instruments
• Includes a generous supply of reagents, sufficient for performing 200 ATP assays using 500 μL sample volumes or 1,000 ATP assays using 100 μL sample volumes (enough for ten 96-well plate assays)
• Facilitates quantitative analysis of ATP levels in a wide range of biological and environmental samples and experimental systems
• Leverages the highly specific and sensitive bioluminescence reaction between luciferin and luciferase in the presence of ATP, providing a reliable and reproducible method for ATP quantification
• Compatible with various luminometers and microplate readers with a luminescence mode, making it a versatile tool for ATP detection in diverse research settings

Principle of the ATP-dependent bioluminescent reaction

The ATP Determination Kit is based on the absolute requirement of luciferase for ATP to produce light. In the presence of Mg²⁺, the luciferase enzyme catalyzes the reaction of luciferin, ATP, and O₂ to form oxyluciferin, AMP, CO₂, pyrophosphate, and light with an emission of approximately 560 nm at pH 7.8. The luminescent signal generated is directly proportional to the amount of ATP, allowing for the measurement of ATP levels. 

Measure ATP levels in solution or cell lysates 

The assay reagents in the ATP Determination Kit are not permeable to live and intact cells, making this assay suitable for measuring extracellular ATP or ATP from samples in solution. To measure intracellular ATP levels for cell health or viability assays, cell lysis is required to release ATP from the cells. Lysis buffer options that are compatible with the luciferase-luciferin reaction include the Pierce Luciferase Cell Lysis Buffer (2X) (Cat. No. 16189) and the 20X cell lysis buffer formulation listed below. The 20X buffer should be diluted 1:20 with water and used immediately for cell lysis prior to running the luciferase ATP assay.

20X lysis buffer:

• 200 mM Tris (pH 7.5)
• 2 M NaCl 
• 20 mM EDTA
• 0.2% Triton X-100 

Comprehensive and versatile ATP assay kit

ATP is a critical molecule found in all living cells and is the energy source for various biological processes, including muscle contraction, nerve impulse propagation, and biochemical synthesis. ATP levels are often measured to determine cellular viability and metabolism, assess mitochondria function and activity, or detect bacterial and microbial contamination. This luciferase-based ATP detection kit provides a versatile and sensitive method for measuring ATP production in various enzymatic reactions, including ATPase and NADPH oxidase, and monitoring ATP release from cells.

The luciferin–luciferase luminescence assay can be used to detect low-level bacterial contamination in biological samples such as blood, milk, and urine, along with environmental samples for testing of water, soil, and sludge. ATP detection has also been used to study the effects of antibiotics on bacterial populations, determine cell proliferation and cytotoxicity in both bacterial and mammalian cells, and distinguish cytostatic versus cytocidal potential of anticancer drugs on malignant cell growth. 

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