ATP Assay Kit

Catalog No. ABIN1000293

Product Details

Target: Adenosine Triphosphate (ATP)

Sample Type: Cell Extracts

Specificity: 0.1 μM

Characteristics: Safe. Non-radioactive assay.
Sensitive and accurate. As low as 0.02 µM ATP or a single cell can be quantified.
Homogeneous and convenient. Mix-incubate-measure type assay. No wash and reagent transfer steps are involved.
Robust and amenable to HTS: Z' factors of > 0.5 are routinely observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems.

Components: Assay Buffer: 10 mL. Substrate: 120 µL. ATP Enzyme: 120 µL. Standard: 100 µL 3 mM.

Application Details

Application Notes: ATP determination in cells and other biological samples.

Comment:

Since the signal of the reaction decreases by ~1% each minute, for most accurate results, care should be taken that the time between adding the Reconstituted Reagent and luminescence reading is the same for all samples and standards.

Protocol: Assays can be carried out in a tube or in a microplate.
1. Standard Curve. Prepare 500 µL 30 µMATP Premix by mixing 5 µL 3 mM Standard and 495 µL distilled water (for cell culture samples dilute ATP in culture media). Dilute standard as shown in the Table. Transfer 10 µL standards into wells of a white opaque 96-well plate. Samples. Use 10 µL sample per well in separate wells. For tissue samples, homogenize 20 mg sample in 200 µL of cold phosphate-buffered saline, spin at 12,000 g for 5 min to pellet any debris. Transfer 1-10 µL supernatant to each well and bring the volume to 10 µL with PBS. Test several doses of the sample and choose the readings that are within the standard curve range for ATP calculation. For suspension cells, transfer 10 µL of the cultured cells (10^3 -10^4 ) into a white opaque 96 well plate. For adherent cells, culture 10^3 -10^4 cells in white opaque microplate. At the time of assay, remove the culture medium immediately before adding 90 µL Reconstituted Reagent (see below).
2. Assay. Bring Assay Buffer and Substrate to room temperature. Thaw enzyme on ice or at 4°C. Fresh Reconstitution is recommended. Store unused reagents including the enzyme at -20°C. For each 96-well, mix 95 µL Assay Buffer with 1 µL Substrate and 1 µL ATP Enzyme. Add 90 µL Reconstituted Reagent to each well. Mix by tapping the plate.
3. Read luminescence on a luminometer within 1 min after adding Reconstituted Reagent.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

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Ponnusamy, Ma, Gong, Pang, Chin, Zhuang: "P2X7 receptors mediate deleterious renal epithelial-fibroblast cross talk." in: American journal of physiology. Renal physiology, Vol. 300, Issue 1, pp. F62-70, (2011) (PubMed).

Aflaki, Radovic, Chandak, Kolb, Eisenberg, Ring, Fertschai, Uellen, Wolinski, Kohlwein, Zechner, Levak-Frank, Sattler, Graier, Malli, Madeo, Kratky: "Triacylglycerol accumulation activates the mitochondrial apoptosis pathway in macrophages." in: The Journal of biological chemistry, Vol. 286, Issue 9, pp. 7418-28, (2011) (PubMed).

Dvoriantchikova, Barakat, Hernandez, Shestopalov, Ivanov: "Liposome-delivered ATP effectively protects the retina against ischemia-reperfusion injury." in: Molecular vision, Vol. 16, pp. 2882-90, (2011) (PubMed).

Khuda-Bukhsh, De, Das, Dutta, Boujedaini: "Analysis of the capability of ultra-highly diluted glucose to increase glucose uptake in arsenite-stressed bacteria Escherichia coli." in: Zhong xi yi jie he xue bao = Journal of Chinese integrative medicine, Vol. 9, Issue 8, pp. 901-12, (2011) (PubMed).

Belleannée, Da Silva, Shum, Brown, Breton: "Role of purinergic signaling pathways in V-ATPase recruitment to apical membrane of acidifying epididymal clear cells." in: American journal of physiology. Cell physiology, Vol. 298, Issue 4, pp. C817-30, (2010) (PubMed).

Chandak, Radovic, Aflaki, Kolb, Buchebner, Fröhlich, Magnes, Sinner, Haemmerle, Zechner, Tabas, Levak-Frank, Kratky: "Efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase." in: The Journal of biological chemistry, Vol. 285, Issue 26, pp. 20192-201, (2010) (PubMed).

Sugimoto, Lin, Lai, Okazaki, Das, Li, Krupnick, Chen, Jeong, Patterson, Kreisel, Gelman: "Apyrase treatment prevents ischemia-reperfusion injury in rat lung isografts." in: The Journal of thoracic and cardiovascular surgery, Vol. 138, Issue 3, pp. 752-9, (2009) (PubMed).

Schwarzer, Fischer, Kim, Barber, Mills, Kurth, Gruenert, Suh, Machen, Illek: "Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells." in: Free radical biology & medicine, Vol. 45, Issue 12, pp. 1653-62, (2008) (PubMed).

Target Details

Target: Adenosine Triphosphate (ATP)

Alternative Name: ATP (ATP Products)

Synonyms: ATP Kit, ATPIB Kit, CAMRQ4 Kit, IB Kit, ML-1 Kit, AI415030 Kit, Atpc1b Kit, Ib Kit, ATPase phospholipid transporting 8A2 Kit, ATPase, aminophospholipid transporter-like, class I, type 8A, member 2 Kit, ATP8A2 Kit, Atp8a2 Kit

Background: Rapid, quantitative, bioluminescent determination of cellular ATP concentration.
Procedure: 10 min.

Adenosine 5'-triphosphate (ATP) is the chemical energy for cellular metabolism and is often referred to as energy currency of the cell. ATP is produced only in living cells during photosynthesis and cellular respiration and consumed in cellular processes including biosynthetic reactions, motility and cell division. It is a key indicator of cellular activity and has been utilized as a measure of cell viability and cytotoxicity in research and drug discovery. This ATP Assay Kit provides a rapid method to measure intracellular ATP. The single working reagent lyses cells to release ATP, which, in the presence of luciferase, immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of intracellular ATP concentration. This non-radioactive, homogeneous cell-based assay is performed in microplates. The reagent is compatible with all liquid handling systems for high-throughput screening applications in 96-well and 384-well plates.