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ADP Assay Kit (Colorimetric/Fluorometric)
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IntroductionThe ADP analysis kit provides a convenient method for measuring ADP levels using colorimetric and/or fluorescence methods. The detection is based on the conversion of ADP into ATP and pyruvate, and the generated pyruvate can be quantitatively determined by colorimetric method (ODmax=570 nm) or fluorescence method (Ex/Em 535/587 nm). The classic method for detecting ADP is to first phosphorylate ADP into ATP and then use the luciferase/luciferin mediated method for detection. However, the luciferase system is unstable, and most laboratories are limited by the lack of bioluminescent instruments. In contrast, this method is simple, sensitive, stable, high-throughput, and widely applicable, and can be detected by traditional enzyme-linked immunosorbent assay (ELISA). This method can detect as low as 1 in biological samples μ M's ADP.ADP is a product of ATP dephosphorylation, which can be re phosphorylated into ATP. Dephosphorylation and rephosphorylation are produced through various phosphatases, phosphorylases, and kinases. ADP exists in platelets and can be released to interact with various purinergic receptors. ADP levels regulate various enzymes involved in intermediate metabolism. The conversion of ADP to ATP mainly occurs in mitochondria and chloroplasts, as well as in the cytoplasm.ComponentsComponents
Storage conditionsADP analysis buffer25 mL-20°CADP probe200 µL-20°C, protect from lightADP converting enzyme1 vial-20°CChromogenic agent1 vial-20°CADP standard1 vial-20°C
Method1. Sample preparation1.1 Sample PreprocessingCell lysate: Collect 1x106 cell samples per well (initial recommended amount).Organizational samples: Collect 10mg/piece of organizational samples (initial recommended amount)Body fluid samples: When collecting plasma or serum, heparin anticoagulation should be used to avoid EDTA and other chelating agents. Normal body fluid samples can be directly used for testing.1.2 Clean cell or tissue samples with cold PBS.1.3 Add 100 µ L of ADP analysis buffer to the sample to resuspend cells or tissues.1.4 Use a pipette to quickly blow the cell sample several times to induce cell lysis.Tissue sample using a Dounce grinder and grind it evenly on ice 10-15 times.1.5 Cell and tissue samples after lysis were placed at 4℃ and centrifuged at 13000g for 5 minutes,1.6 Collect the supernatant and transfer it to a new centrifuge tube.1.7 Temporarily store on ice for testing.2.Preparation of reagentsEach reagent is centrifuged at low speed before opening the tube.2.1 ADP analysis buffer should be equilibrated to room temperature before use.2.2 ADP standard: Dissolve in 200 µ L dH2O to prepare 10mM ADP standard stock solution, and store it temporarily on ice.2.3 ADP probe: Thaw in a 37 ℃ water bath for 1-5 minutes before use.2.4 ADP Converting Enzyme: Dissolve 220 µ L of ADP in buffer before use and temporarily store on ice,2.5 Chromogenic agent: Dissolve 220 µ L of ADP analysis buffer before use and temporarily store on ice,3. Preparation of gradient diluted standard solutionA new standard solution must be prepared for each test, and the standard solution must not be stored after use.Gradient dilution is performed using colorimetric methods within the range of [2-10 nmol/well] or fluorescence methods within the range of [0.2-1 nmol/well].3.1 Standard solution for colorimetric method: Take 10 µ L of ADP standard stock solution (10mM) and add 90 µ L of dH2O to prepare 1 mM of ADP standard solution, mix well.Take 0, 6, 12, 18, 24, and 30 µ L of ADP standard solution (1mM) and add it to the series of wells. Add ADP analysis buffer and make up to 50 µL/well to prepare standard solutions of 0, 2, 4, 6, 8, and 10 nmol/well.3.2 Standard solution for fluorescence method: Dilute the ADP standard (1mM) to 0.1mM using ADP analysis buffer, and then prepare gradient standard solutions of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 nmol/well according to the above method.Note: The detection sensitivity of fluorescence analysis method is 10-100 times higher than that of colorimetric method.4. Prepare ADP reaction solutionPrepare the required ADP reaction solution according to the requirement of 50 µL for each detection well.The preparation ratio of each component in 50 µL is shown in the table below:Colorimetric methodReaction solution/blank (µL)Fluorescence methodReaction solution/blank (µL)ADP analysis buffer44 / 4645.8 / 47.8ADP probe2 / 20.2 / 0.2ADP converting enzyme2 / 02 / 0Chromogenic agent2 / 22 / 25. Add 1-50 µL of sample to each well in the sample well. If it is less than 50 µL, use the analysis buffer to supplement it to 50 µL/well.Note: Sample background control wells can be set.6. Add 50 µL of reaction solution to each well in the standard well, sample well, and sample background control well. Add 50 µ L of blank reaction solution to the blank control well. -7. Incubate the 96 well plate at room temperature in the dark for 30 minutes.The colorimetric method was used to detect at OD 570 nm by the enzyme-linked immunosorbent assay (ELISA) reader,Fluorescence assay was performed using a fluorescence enzyme-linked immunosorbent assay (ELISA) with Ex/Em=535/587 nm. Testing needs to be completed within 2 hours.8. Calculate ADP contentIf the reading of the sample exceeds the standard range, the analysis buffer should be used for dilution and retesting.8.1 Calculate the average reading of each compound well in the standard, control, and sample.8.2 Subtract the average value of the blank well (Standard # 1) from all standard, control, and sample readings. This is the corrected absorbance value/relative fluorescence value. Subtract the background control reading from the sample reading.8.3 Construct a standard curve by comparing the calibration values of each standard well with the concentration of ADP, and fit the trend line equation.8.4 Apply the corrected absorbance/relative fluorescence values of the sample to the standard curve to obtain the amount of ADP (B) in the sample well.8.5 The ADP concentration (nmol/µ L or µ mol/mL or mM) in the sample is calculated as:ADP concentration=(B/V) DIn the formula:B=The amount of ADP in the sample well calculated based on the standard curve (nmol or mM).V=Volume of sample added to the sample well (µ L).D=dilution factor of the sample.ADP molecular weight=427.2 g/mol
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