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.
Key Features:Safe. Non-radioactive assay
Sensitive and accurate. As low as 0.02uM 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.
Applications:ADP determination in cells and other biological samples.
Kit Contents:Assay Buffer: 10ml Substrate: 120ulCosubstrate: 120ul ATP Enzyme: 120ulADP Enzyme: 120ul Standard: 100ul 3mM ADPStorage conditions: store all reagents at -20°C. This product is shipped on dry ice. Shelf life: 6 months after receipt
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Assay Procedure: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 500ul 30uM ADP Premix by mixing 5ul 3mM Standard and 495ul distilled water (for cell culture samples dilute ADP in culture media). Dilute standard as follows. Transfer 10ul standards into wells of a white opaque 96-well plate
No Premix+H2O/media Vol (ul) ADP (uM)1 50ul+0ul 50 302 40ul+10ul 50 243 30ul+20ul 50 184 20ul+30ul 50 125 15ul+35ul 50 96 10ul+40ul 50 67 5ul+45ul 50 38 0ul+50ul 50 0Samples. Use 10ul sample per well in separate wells. For tissue samples, homogenize 20 mg sample in 200ul of cold phosphate-buffered saline, spin at 12,000 g for 5 min to pellet any debris. Transfer 1-10ul supernatant to each well and bring the volume to 10ul 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 10ul of the cultured cells (103-104) into a white opaque 96 well plate. For adherent cells, culture 103-104 cells in white opaque microplate. At the time of assay, remove the culture medium immediately before adding 90ul 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 95ul Assay Buffer with 1ul Substrate, 1ul Cosubstrate and 1ul ATP Enzyme. Add 90ul 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 5ul dH2O with 1ul ADP Enzyme. Immediately following reading RLU A, add 5ul 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
4. Calculation: of ADP Concentration. Subtract RLU A from RLU B for standards and samples. Plot the dRLU versus ADP concentration for the standards. From the slope of this plot, the Sample ADP concentration can be computed with the following equation: [ADP]sample (uM)=(RLU B)sample–(RLU A)sampleSlope
General Considerations:Signal stability. 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.
