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Protokoll
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A. Reagent Preparations: Dilute the 10X cAMP Assay Buffer to 1X Assay Buffer with MilliQ water. Store at 4 o C. Reconstitute the Standard cAMP (pellet may not be visible) in 1 ml of 0.1M HCl (not provided), vertex for 10 seconds to generate 10 pmol/ μ l cAMP standard stock solution. Dilute the rabbit anti-cAMP pAb and cAMP-HRP each with 1.1 ml of the 1X Assay Buffer as stock solutions, keep frozen. Unused Protein G coated strips can be kept at –20 o C with descants, stable for up to 1 month after opening. The kit should be stored at –20 o C. After opening and reconstitution, components can be stored as instructed in the kit contents above, stable for 1 to 2 months. B. General Consideration: Esterases may degrade cAMP. Therefore, prepare samples in 0.1N HCl to inactivate esterase, and store at -80 o C. Dilute your samples to ~0.1- 10pmol/5 μ l cAMP range. Urine and tissue culture supernatant can be diluted in 10% 1M HCl and assayed directly. Plasma, serum, whole blood, and tissue homogenates often contain phosphodiesterases and large amount of immunoglobulins (Igs) which may interfere with the assay. However, diluting these samples with 0.1M HCl can generally inactivate phosphodiesterases and lower the concentration of Igs, making the samples suitable for the assay. Both phospho-diesterases and Igs can also be removed by 5% TCA precipitation or 10 kD molecular weight cut off micro centrifuge filters (BioVision Cat #1997-25). To determine whether interference is presence in your sample, you may make two different dilutions. If the two different dilutions of sample show good correlation in the final calculated cAMP concentrations, purification is not required. If you do not see good correlation of the different dilutions, purification by TCA precipitation or 10 Kd molecular weight cut off microcentrifuge filters to remove any enzymes from samples. Organic solvents in samples may interfere with the assay, which may need to be removed prior to the assay. C. Sample Preparation: Urine, Plasma and Culture Medium Samples: Urine and plasma may be tested directly with 1:20 to 1:100 dilutions in 0.1M HCl. Culture medium can also be tested with 1:10 to 1:200 dilutions in 0.1M HCl. (Note: RPMI medium may contain >350 fmol/ μ l cAMP). Cell Samples: Aspirate medium. Add 1 ml of 0.1M HCl for every 35 cm 2 of surface area. Incubate at room temperature for 20 minutes. Scrape cells off the surface with a cell scraper. Dissociate sample by pipetting up and down until suspension is homogeneous. Transfer to a centrifuge tube and centrifuge at top speed for 10 min. The supernatant can be assayed directly. Protein concentration >1 mg/ml is recommended for reproducible results. Tissue Samples: Cyclic nucleotides may be metabolized quickly in tissue, so it is important to rapidly freeze tissues after collection (e.g., using liquid nitrogen). Weigh the frozen tissue and add 5-10 volume of 0.1M HCl. Homogenize the sample on ice using a Polytron-type homogenizer. Spin at top speed for 5 min and collect the supernatant. The supernatant may be assayed directly. D. cAMP Assay: The procedure described here includes an acetylation step which makes the cAMP assay much more sensitive and avoid the interferences of many components in samples. However, for routine assay of the well known samples, non-acetylation procedure may also be used, just skip the acetylation steps (Step 7 and 8). Prepare cAMP Standard Curve and Samples: 1. Dilute 200 μ l of the 10 pmol/ μ l standard cAMP stock with 800 μ l of 0.1M HCl to generate 2 pmol/ μ l cAMP working solution. The diluted cAMP should be fresh, and used within 1 hour. 2. Label 8 microcentrifuge tubes, 200, 100, 50, 25, 12.5, 6.25, 3.12, 0 pmol. 3. Add 200 μ l of the 2 pmol/ μ l cAMP standard into the tube labeled 200 pmol tube (amount enough for 20 assays). Add100 μ l 0.1M HCl into the rest of tubes. 4. Transfer 100 μ l of the 2 pmol/ μ l standard cAMP from the tube labeled 200 pmol tube to the labeled 100 pmol tube, mix, then transfer 100 μ l into the labeled 50 pmol tube. Continue the serial dilution by transfer 100 μ l to 25, 12.5, 6.25, 3.1 pmol tubes. The diluted cAMP should be used within 1 hour. 5. Add 100 μ l test samples to new labeled tubes. We suggest performing different dilutions for each sample with 0.1M HCl to ensure the sample readings are within standard curve. 6. Add 50 μ l of Neutralizing Buffer to each tube (all standards cAMP and testing samples). 7. Prepare Acetylating Reagent Mix: Mix 1 volume of Acetylating Reagent A (Violet cap) with two volumes of Acetylating Reagent B (Amber cap) in a microtube. Prepare enough for the experiment (need 5 μ l each sample and standard tubes). Use within 1 hour. 8. Add 5 μ l of the Acetylating Reagent Mix directly into each test solution (all standards and samples), IMMEDIATELY vertex 2-3 seconds following each addition without delay, one tube at a time, and incubate at room temperature for 10 min to acetylate cAMP. 9. Add 845 μ l 1X Assay Buffer into each tube to dilute the acetylation reagents, mix well. The acetylated standard and samples are ready for quantification.(If cAMP in your samples are very low, the acetylation reagents can be dried after step 8, without dilution step 9 to minimize the volume increase).Then reconstituted in a 50 -100 μ l volume of Assay Buffer. BioVision rev. 09/09
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Applikationshinweise
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Note: RPMI medium may contain >350 fmol/ μ l cAMP). Cell Samples: Aspirate medium. Add 1 ml of 0.1M HCl for every 35 cm 2 of surface area. Incubate at room temperature for 20 minutes. Scrape cells off the surface with a cell scraper. Dissociate sample by pipetting up and down until suspension is homogeneous. Transfer to a centrifuge tube and centrifuge at top speed for 10 min. The supernatant can be assayed directly. Protein concentration >1 mg/ml is recommended for reproducible results. Tissue Samples: Cyclic nucleotides may be metabolized quickly in tissue, so it is important to rapidly freeze tissues after collection (e.g., using liquid nitrogen). Weigh the frozen tissue and add 5-10 volume of 0.1M HCl. Homogenize the sample on ice using a Polytron-type homogenizer. Spin at top speed for 5 min and collect the supernatant. The supernatant may be assayed directly. D. cAMP Assay: The procedure described here includes an acetylation step which makes the cAMP assay much more sensitive and avoid the interferences of many components in samples. However, for routine assay of the well known samples, non-acetylation procedure may also be used, just skip the acetylation steps (Step 7 and 8). Prepare cAMP Standard Curve and Samples: 1. Dilute 200 μ l of the 10 pmol/ μ l standard cAMP stock with 800 μ l of 0.1M HCl to generate 2 pmol/ μ l cAMP working solution. The diluted cAMP should be fresh, and used within 1 hour. 2. Label 8 microcentrifuge tubes, 200, 100, 50, 25, 12.5, 6.25, 3.12, 0 pmol. 3. Add 200 μ l of the 2 pmol/ μ l cAMP standard into the tube labeled 200 pmol tube (amount enough for 20 assays). Add100 μ l 0.1M HCl into the rest of tubes. 4. Transfer 100 μ l of the 2 pmol/ μ l standard cAMP from the tube labeled 200 pmol tube to the labeled 100 pmol tube, mix, then transfer 100 μ l into the labeled 50 pmol tube. Continue the serial dilution by transfer 100 μ l to 25, 12.5, 6.25, 3.1 pmol tubes. The diluted cAMP should be used within 1 hour. 5. Add 100 μ l test samples to new labeled tubes. We suggest performing different dilutions for each sample with 0.1M HCl to ensure the sample readings are within standard curve. 6. Add 50 μ l of Neutralizing Buffer to each tube (all standards cAMP and testing samples). 7. Prepare Acetylating Reagent Mix: Mix 1 volume of Acetylating Reagent A (Violet cap) with two volumes of Acetylating Reagent B (Amber cap) in a microtube. Prepare enough for the experiment (need 5 μ l each sample and standard tubes). Use within 1 hour. 8. Add 5 μ l of the Acetylating Reagent Mix directly into each test solution (all standards and samples), IMMEDIATELY vertex 2-3 seconds following each addition without delay, one tube at a time, and incubate at room temperature for 10 min to acetylate cAMP. 9. Add 845 μ l 1X Assay Buffer into each tube to dilute the acetylation reagents, mix well. The acetylated standard and samples are ready for quantification.(If cAMP in your samples are very low, the acetylation reagents can be dried after step 8, without dilution step 9 to minimize the volume increase).Then reconstituted in a 50 -100 μ l volume of Assay Buffer. BioVision rev. 09/09 BioVision Research Products Tel: 650-428-0236 • Fax: 650-428-0336 980 Linda Vista Avenue, Mountain View, CA 94043 USA www.biovision.com • tech@biovision.com For research use only Quantification cAMP: 1. Add 50 μ l of the acetylated Standard cAMP and test samples from Step 9 to the Protein G coated 96-well plate. 2. Add 10 μ l of the reconstituted cAMP antibody to the test sample and standard, except the 0 pmol cAMP well as reagent background . Incubate for 1 hour at room temperature with agitation. It is recommended to use repeating pipette to minimize pipette errors. 3. Add 10 μ l of cAMP-HRP to each well, incubate for 1 hr at room temperature with agitation. 4. Wash 5 times with 200 μ l 1X Assay Buffer each wash. 5. Add 100 μ l of HRP developer and develop for 1 hour at room temperature with agitation. 6. Stop the reaction by adding 100 μ l of 1M HCl (not provided) to each well (sample color should change from blue to yellow). 7. Read the plate at OD 450 nm. The OD450 nm reading varies significantly among experiments depend on lot numbers, kit storage and experiment conditions. Therefore, samples and standard curve must be performed at the same time and using the same kit reagents. 8. Calculations: Subtract OD 450nm background reading (0 pmol cAMP) from all samples and standards reading. Plot standard curve OD450 nm vs cAMP amount. Apply OD readings from samples to the standard curve. Calculate amount of cAMP in samples: cAMP Concentration = Sa/Sv (pmol/ml, or nM) Where Sa is cAMP amount (pmol) from standard curve. Sv is sample volume (ml) added into the assay wells (50 μ l acetylated sample contains 5 μ l (0.005 ml) original sample before acetylation step). cAMP Standard Curve: The assay was performed following the kit protocol. VI.
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