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Beschreibung
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ADP is a product of ATP dephosphorylation and it can be rephosphorylated to ATP. De-phosphorylation and rephosphorylation occur via various phosphatases, phosphorylases and kinases. ADP is stored in platelets and can be released to interact with a variety of purinergic receptors. ADP levels regulate several enzymes involved in intermediary metabolism. ADP conversion to ATP primarily occurs within the mitochondrion and chloroplast although several such processes occur in the cytoplasm. Conventionally, ADP levels are measured by luciferase/luciferin mediated assays after ADP is converted to ATP. However, the luciferase system is unstable and luminescence equipment is not generally available in most laboratories. BioVision's newly designed ADP Assay Kit provides an convenient colorimetric and fluorometric means to measure ADP level. In the assay, ADP is converted to ATP and pyruvate. The generated pyruvate can be quantified by colorimetric (lambda max = 570 nm) or fluorometric method (Ex/Em 535/587 nm). The assay is simple, sensitive, stable and high-throughput adaptable. The assay can detect as low as 1 µM ADP in biological samples. Contents: III. Storage and Handling: Store kit at -20 o C, protect from light. Warm ADP Assay Buffer to room temperature prior to use. Briefly centrifuge all vials prior to opening. Read the entire protocol before performing the assay. IV. Reagent Preparation and Storage Conditions: ADP Probe: Dissolve with 220 µl of DMSO (provided, warm up >18 o C to become liquid) before use. Mix well, store at -20 o C, protect from light and moisture. Use within two months. ADP Converter and ADP Developer Mix: Dissolve with 220 µl ADP Assay Buffer separately. Pipette up and down to dissolve. Store at -20 o C. Use within two months. ADP Standard: Dissolve in 100 µl dH2O to generate 10 mM stock solution. Keep cold while in use. Store at -20°C. V. ADP Assay Protocol: 1. Standard Curve Preparations: For the Colorimetric Assay: Dilute the ADP Standard to 1 nmol/ µl by adding 10 µl of the 10 mM Standard to 90 µl of ADP Assay Buffer, mix well. Add 0, 2, 4, 6, 8, 10 µl into a series of standards wells. Adjust volume to 50 µl/well with ADP Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of ADP Standard. For the Fluorometric Assay: Dilute the ADP Standard to 0.1 nmol/ µl (the fluorometric assay is 10 to 100 fold more sensitive than the colorimetric assay). Add 0, 2, 4, 6, 8, 10 µl into a series of standards wells. Adjust volume to 50 µl/well with Assay Buffer to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well of ADP standard. 2. Sample Preparation: Liquid samples can be measured directly. Tissue (10 mg) or cells (10 6 ) can be homogenized in 100 µl of ADP Assay Buffer, spin at 12,000 X g for 5 min to remove insoluble materials. Add 1-50 µl sample to each well in a 96-well plate, bring the volume to 50 µl with Assay Buffer. 3. Intracellular ADP level is usually in the range of 0.1-3 mM. We suggest testing several doses of your sample to ensure the readings are within the standard curve range. 4. ADP Reaction Mix: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µl Reaction Mix containing: Colorimetric Assay Fluorometric Assay ADP Assay Buffer 44 µl 45.8 µl ADP Probe 2 µl 0.2 µl * ADP Converter** 2 µl 2 µl ADP Developer 2 µl 2 µl Notes: *For the fluorometric assay, use 1/10 of ADP probe to reduce fluorescence background. **Pyruvate generates background. If significant amount of pyruvate is suspected in your samples, a sample pyruvate background control need to be performed by replacing the ADP Converter with 2 µl of assay buffer. Then follow the same protocol as the sample. In the absence of ADP converter, the assay detects only pyruvate, not ADP. The pyruvate background reading can be subtracted from the ADP readings. 5. Add 50 µl of the Reaction Mix to each well containing the ADP Standard and test samples. Incubate at room temperature for 30 minutes, protect from light. 6. Measure O.D. 570 nm for colorimetric assay or Ex/Em 535/587 nm for fluorometric assay. 7. Calculation: Subtracting the 0 ADP control from all standard and sample readings (
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