ADP Assay Kit

Catalog No. ABIN1000287

Product Details

Target: ADP (Adenosine Diphosphate (ADP))

Sample Type: Cell Extracts

Specificity: 0.1 μM ADP

Characteristics: Safe. Non-radioactive assay.
Sensitive and accurate. As low as 0.02 µM ADP 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 and above are routinely observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.

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

Application Details

Application Notes: ADP 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 96-well plate. For consistency, it is recommended that the time between the two luminescence measurements be the same for all samples.
1. Standard Curve. Prepare 500 µL 30 µMADP Premix by mixing 5 µL 3 mM Standard and 495 µL distilled water (for cell culture samples dilute ADP in culture media). 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 ADP 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 ATP Reagent (see below).
2. ATP Reaction. Bring Assay Buffer, Substrate and Cosubstrate to room temperature. Thaw enzyme on ice or at 4°C. Fresh Reconstitution is recommended. Store unused reagents including the enzyme at -20°C. ATP Reagent. For each 96-well, mix 95 µL Assay Buffer with 1 µL Substrate, 1 µL Cosubstrate and 1 µL ATP Enzyme. Add 90 µL ATP Reagent to each well and mix by tapping the plate. After 10 min, read luminescence (RLU A) on a luminometer.
3. ADP Assay. Prepare ADP Reagent: for each 96-well, mix 5 µL dH2O with 1 µL ADP Enzyme. Immediately following reading RLU A, add 5 µL ADP Reagent to each well and mix by tapping the plate or pipetting up and down. Incubate for 2 minutes at room temperature. Read luminescence (RLU B) on a luminometer.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Helms, Marvel, Zhao, Stahle, Vest, Kato, Lee, Christ, Gladwin, Hantgan, Kim-Shapiro: "Mechanisms of hemolysis-associated platelet activation." in: Journal of thrombosis and haemostasis : JTH, Vol. 11, Issue 12, pp. 2148-54, (2014) (PubMed).

Chatterjee, Sparks: "Hepatic lipase release is inhibited by a purinergic induction of autophagy." in: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, Vol. 33, Issue 4, pp. 883-94, (2014) (PubMed).

Cao, Li, Qin, Li, Zhang, Wang, Hu, Fang, Gao, Li, Sun, Xiong, Gao, Zhu: "Astrocytic adenosine 5'-triphosphate release regulates the proliferation of neural stem cells in the adult hippocampus." in: Stem cells (Dayton, Ohio), Vol. 31, Issue 8, pp. 1633-43, (2013) (PubMed).

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).

Vacirca, Barbati, Scazzocchio, Masella, Rosano, Malorni, Ortona: "Anti-ATP synthase autoantibodies from patients with Alzheimer's disease reduce extracellular HDL level." in: Journal of Alzheimer's disease : JAD, Vol. 26, Issue 3, pp. 441-5, (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).

Target Details

Target: ADP (Adenosine Diphosphate (ADP))

Alternative Name: ADP (ADP Products)

Synonyms: 6720489L24Rik Kit, 9330209L04Rik Kit, Adp Kit, Mamdc1 Kit, RNSVSP4U Kit, SVSIV1 Kit, Svp4 Kit, ADP Kit, DCAF9 Kit, MAM domain containing glycosylphosphatidylinositol anchor 2 Kit, seminal vesicle secretory protein 4 Kit, WD and tetratricopeptide repeats 1 Kit, Mdga2 Kit, Svs4 Kit, WDTC1 Kit

Background: Rapid, quantitative, bioluminescent determination of ADP concentration.
Procedure: 20 min.

This TM ADP Assay Kit provides a rapid method to measure ADP levels. The assay involves two steps. In the first step, the working reagent lyses cells to release ATP and ADP. In the presence of luciferase, ATP immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of intracellular ATP concentration. Luciferase ATP + D-luciferin + O2 oxyluciferin + AMP + PPi + CO2 + light In the second step, the ADP is converted to ATP through an enzyme reaction. This newly formed ATP then reacts with the D-luciferin as in the first step. The second light intensity measured represents the total ADP and ATP concentration in the sample. This non-radioactive, homogeneous cell-based assay is performed in microplates. The reagent is compatible with all culture media and with all liquid handling systems for high-throughput screening applications in 96-well and 384-well plates.