Living things require a continual input of free energy for three major purposes: the performance of mechanical work in muscle contraction and other cellular movements, the active transport of molecules and ions, and the synthesis of macromolecules and other biomolecules from simple precursors.
The free energy used in these processes is derived from the surroundings and maintains an organism in a state that is far from equilibrium. In most processes, this special carrier of free energy is adenosine triphosphate (ATP), an energy-rich molecule. Its triphosphate unit contains two phosphoanhydride bonds. The turnover of ATP is very high. Motion, active transport, signal amplification, and biosynthesis (as is needed for cell proliferation) can occur only if ATP is continuously regenerated from ADP. Therefore, measurement of ATP can serve as a marker for cell proliferation.
The determination of ATP using bioluminescence is a well established technique. It uses the ATP dependency of the light-emitting, luciferase-catalyzed oxidation of luciferin for the measurement of extremely low concentrations of ATP.
The ATP Bioluminescence Assay Kit HS II is specifically developed for the high-sensitivity detection of ATP. Due to the high concentration of luciferase in the assay, the reaction exhibits a peak kinetic. If ATP determinations are performed to obtain kinetic data of the enzymes involved (e.g., for metabolic studies or if coupled enzymatic assays are applied), the use of the ATP Bioluminescence Assay Kit CLS II (generating a constant light signal) is highly recommended.
Highly sensitive and quantitative determination of ATP. Contains a cell lysis reagent and can be applied for the detection of microbial contamination. The kit is well suited for use in tube luminometers and microplate-format luminometers. The cell lysis reagent exhibits good lysis efficiency with respect to a variety of eukaryotic and prokaryotic cells.
For a time constant light signal, use the ATP Bioluminescence Assay Kit CLS II.
For maximum sensitivity, the sample ATP must be in a minimum volume, and the luciferase reagent must not be diluted.
The luciferase from Photinus pyralis (American firefly) catalyzes the following reaction:
ATP + D-luciferin + O2? oxyluciferin + PPi + AMP + CO2 + light.
The quantum yield for this reaction is about 90%. The resulting green light has an emission maximum at 562 nm.
The Michaelis equation has the following form:
light intensity = (Vmax x CATP)/(Km + CATP).
At low ATP concentrations (CATPm), the formula is simplified to light intensity = Vmax x CATP/Km.
From this equation, it becomes obvious that the light output is directly proportional to the ATP concentration (CATP), and is dependent on the amount of luciferase (Vmax) present in the assay.
1 kit containing 4 components.
Working concentration: 10-5 to 10-12 M ATP
Working solution: Preparation of Working Solutions
Dissolve the whole content of one vial 1 by carefully adding 10 ml of dilution buffer from vial 4. Incubate for 5 minutes at 0 to 4 °C without stirring or shaking. Mix for a homogeneous solution by carefully rotating the bottle. Do not shake!
Stability: Set up a standard curve each day, because a slight loss of luciferase activity may occur during this time (approx. 20% after storage for 5 days at 2 to 8 °C).
Information Note: Each bottle contains approx. 10 mg ATP (> 98% purity; Mr 605.2). The exact amount of ATP is determined individually for each lot as indicated on the label.
Dissolve the content of one vial 2 by addition of the appropriate volume of dilution buffer to get a final concentration of 10 mg/ml or 16.5 mM, respectively (e.g., 960 μl to 9.60 mg ATP).
The ATP standard curve is prepared by serial dilutions of one ATP standard with dilution buffer.
Cell Lysis Reagent
The cell lysis reagent is ready-to-use.
Important Note: Avoid contamination by microorganisms or ATP. The use of autoclaved or heat sterilized labware is recommended.
The dilution buffer supplied with the kit is essentially free of ATP and microbial contaminations. Use this buffer for reconstitution and dilution of the kit reagents. It can also be used as diluent for samples.
Important Note: Avoid contamination and use autoclaved or heat sterilized labware.
Storage conditions (working solution): Luciferase Reagent
The reagent is stable for one day at 15 to 25 °C or for one week when stored at 0 to 4 °C.
Reconstituted Luciferase Reagent may be stored frozen at -15 to -25 °C for longer periods of time.
Important Note: Each freeze/thaw-cycle reduces luciferase activity, depending on the freezing conditions (shock freezing is most effective). For best results, avoid repeated freezing and thawing.
When stored at 2 to 8 °C, the solution is stable for one week (< 5% degradation).
When stored at 15 to 25 °C, the solution is stable for at least 4 weeks (< 5% degradation).
When stored on ice, diluted ATP-standards are stable for 8 hours.
Cell Lysis Reagent
The reagent is stable at 2 to 8 °C.
Store at 2 to 8 °C.
For life science research only. Not for use in diagnostic procedures.