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Cat. No.: EATPAK-200 (for 200T)

Description

Enhanced ATP Assay Kit can be used to detect ATP (adenosine 5'-triphosphate) levels in common solutions, cells, or tissues. Sample preparation can be completed with one-step lysis of cells and tissue samples, and the detection sensitivity reaches as high as 0.1 nM. The chemiluminescence can remain stable for up to 30 minutes. Several indicators of this kit surpass similar products on the market, and the obtained samples can also be used for Western blot analysis.

ATP, as the most important energy molecule, plays a vital role in various physiological and pathological processes within cells. Changes in ATP levels can affect cellular functions. Typically, ATP levels decrease during apoptosis, necrosis, or when cells are in certain toxic states. Conversely, stimuli such as high glucose can increase intracellular ATP levels in some cells. A decline in ATP levels often indicates impaired or diminished mitochondrial function, and during apoptosis, this decrease in ATP levels usually coincides with a drop in mitochondrial membrane potential.

This kit is developed based on the requirement of firefly luciferase (also known as luciferase) for ATP to provide energy when catalyzing the luminescence of luciferin. When firefly luciferase and luciferin are both in excess, the luminescence generated is proportional to the concentration of ATP within a certain range. This allows for the highly sensitive detection of ATP concentration in a solution.

Components

• ATP Detection Reagent
• ATP Detection Reagent Diluent
• ATP Standard Solution (0.5 mM)
• ATP Detection Lysis Buffer

Features

• Sample preparation is simple. The kit provides an ATP detection lysis buffer that can be used for lysing cells and tissues. After simple lysis, it can be used for ATP detection without the need for tedious procedures such as perchloric acid or trichloroacetic acid (TCA) extraction or boiling the sample after lysis.
• The sensitivity is high, and the linear range is wide, with effective detection between 0.1 nM and 10 μM. This kit can detect ATP concentrations as low as 0.1 nM with a sample volume of 100 microliters. Typically, ATP concentrations in conventional cell or tissue lysates range from 0.1 to 1 μM, and the intracellular ATP level in some common cells is about 10 nmol/mg protein. Additionally, the detection concentration range of this kit is very wide, with an upper detection limit of up to 10 μM, and it can form a good standard curve within the range of 0.1 nM to 10 μM.
• Readings are stable. The kit is specially optimized to ensure that the chemiluminescence is very stable during ATP detection. The detection results of the ATP standard curve show that there is no significant decrease in chemiluminescence within the first 10 minutes after the reaction starts, and the decrease in chemiluminescence does not exceed 10% within the first 30 minutes after the reaction starts.
• The prepared sample has good compatibility. The cell or tissue samples obtained by lysis with the ATP detection lysis buffer in this kit can be used not only for ATP detection but also for protein concentration measurement, SDS-PAGE, or Western blot analysis of some easily soluble proteins.
• It is convenient and fast to use. Typically, 10-20 samples can be measured within 30-60 minutes.

Storage

Store at -20°C for six months. Store at -70°C for one year. ATP Detection Reagent should be stored protected from light.

Precautions

• The ATP Detection Reagent contains luciferase, which can gradually lose activity with repeated freeze-thaw cycles. Although tests have shown that repeated freeze-thaw cycles up to five times do not significantly affect the detection performance, it is recommended that users limit freeze-thaw cycles to no more than three times for optimal results. After diluting the ATP Detection Reagent into the ATP Detection Working Solution, it is best to use it all at once and avoid refreezing for later use.
• ATP, especially in lysed samples, is unstable at room temperature and should be handled at 4°C or on ice. ATP can remain stable on ice for up to 6 hours.
• This kit requires the use of a luminometer, a chemiluminescence instrument used for detecting luciferase reporter genes. If a luminometer is not available, a liquid scintillation counter can also be used. The effectiveness of a liquid scintillation counter depends on its detection sensitivity and precision.
• When using a multifunctional microplate reader capable of detecting chemiluminescence, it is recommended to use 96-well white or black plates that are opaque between wells. If using a standard clear 96-well plate, special care must be taken to use spacer wells between detection wells to reduce interference from adjacent wells. For clear 96-well plates, a luminescent well can increase the relative light units (RLU) of adjacent wells by about 10-20% and increase the RLU of wells separated by one well by about 1-4%. For the same sample, the chemiluminescence readings of a 96-well white plate with an opaque bottom can be approximately 5-10 times higher than those of a clear 96-well plate and about 3 times higher than those of a white plate with a transparent bottom (actual measurements may vary depending on the 96-well plate, detection instrument, and sample).
• The ATP Detection Lysis Buffer provided in this kit can effectively lyse and release ATP from common cultured cells and tissues. For some special tissues or samples, if the detected ATP level is significantly lower than expected, a portion of the sample can be boiled for 2 minutes after lysis and before centrifugation to fully release ATP. Proteins in the boiled samples will denature and precipitate in the subsequent centrifugation step, so boiled samples cannot be used for protein concentration measurement, SDS-PAGE, or Western blot analysis. Remaining samples can be used for protein concentration measurement, SDS-PAGE, and Western blot analysis.
• This product is intended for scientific research use by professionals only and is not for use in clinical diagnosis or treatment, food, or drugs, nor should it be stored in a regular household.
• For your safety and health, please wear a lab coat and disposable gloves while handling.