|
Protocol
|
A. Reagent Preparations: Dilute the 10X cGMP Assay Buffer to 1X Assay Buffer with MilliQ water. Store at 4 o C. Reconstitute the Standard cGMP (pellet may not be visible) in 1 ml of 0.1M HCl (not provided), vertex for 10 seconds to generate 10 pmol/ μ l cGMP stock standard solution. Reconstitute rabbit anti-cGMP pAb and cGMP-HRP each with 1.1 ml of the 1X Assay Buffer as stock solutions. Unused well strips can be kept at –20 o C with the desiccants, stable for up to 1 month. The kit should be stored at –20 o C. After reconstitution, some components may be stored at 4 o C as instructed above, stable for up to 1- 2 months. B. General Consideration: cGMP samples in 0.1 M HCl (final concentration) is stable and can be used directly in the assay. Make dilutions of your sample with 0.1 M HCl to the range of 1-200 pmol/ml. Plasma, serum, whole blood, and tissue homogenates often contain phosphodiesterases and large amount of immunoglobulins (Igs) which may interfere with the assay. However, preparing samples in 0.1 M 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 by using 10 Kd molecular weight cut off microcentrifuge filters (BioVision Cat.# 10KC-20). 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 cGMP concentrations, purification is not required. If you do not see good correlation of the different dilutions, deproteinize the sample by using TCA or 10 Kd molecular cut off microcentrifuge filters. Some organic solvents may interfere with the assay and may need to be removed prior to the assay. C. Sample Preparation: Urine, Plasma and Culture Media: Urine, plasma, and culture media may be tested directly after adding 1/10 volume of 1M HCl, and remove precipitates if occur. Culture Cells: Collect cells by centrifugation. 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. cGMP Prepare cGMP Standard Curve and Samples: 1. Add 200 μ l of the 10 pmol/ μ l standard cGMP stock into 800 μ l of 0.1M HCl to generate 2 pmol/ μ l cGMP working solution. The diluted cGMP should be used within 1 hour. 2. Label 10 microcentrifuge tubes, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.039, 0 pmol/50 μ l. 3. Add 200 μ l of the 2 pmol/ μ l cGMP into the tube labeled 10 pmol (enough for 20 tests) , add100 μ l 0.1M HCl into the rest of tubes. 4. Transfer 100 μ l from the 10 pmol tube into the labeled 5 pmol tube, mix. Continue the serial dilution by transfer 100 μ l from the 5 pmol tube to 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.039 pmol tubes. Discard the 100 μ l mixture from the last 0.039 pmol tube. The diluted cGMP should be used within 1 hour. 5. Label new tubes for test samples, add 100 μ l each test sample per tube. We suggest using different dilutions for each sample (dilute with 0.1M HCl). 6. Add 50 μ l of Neutralizing Buffer to each tube (both standard cGMP and testing samples) to neutralize the HCl in the samples. 7. Prepare Acetylating Reagent Mix (Note: 5 μ l is needed for each assay): Mix 1 volume of Acetylating Reagent A (Violet cap) with two volumes of Acetylating Reagent B (Amber cap) in a microtube. Prepare just enough for the experiment. Use within 1 hour. 8. Add 5 μ l of the Acetylating Reagent Mix directly into each test solution (both standard and sample), IMMEDIATELY vertex 2-3 seconds following each addition, and incubate at room temperature for 10 min. 9. Add 845 μ l 1X Assay Buffer into each tube, mix well. Use for below quantification. Note: The acetylation step improves the assy sensitivity significantly and avoid the interferences of many components in unpurified samples. Component K372-100 Color Code Storage Part 100 assays Cap Color Temperature Number 10X cGMP Assay Buffer 25 ml WM +4 o C K372-100-1 Standard cGMP (10 nmol) 1 vial Yellow -20 o C K372-100-2 Neutralizing Buffer 7.5 ml NM +4 o C K372-100-3 Acetylating Reagent A 0.75 ml Violet +4 o C K372-100-4 Acetylating Reagent B 1.5 ml Brown +4 o C K372-100-5 Anti-cGMP pAb/BSA 1 vial Red -20 o C K372-100-6 cGMP-HRP/BSA 1 vial Green -20 o C K372-100-7 HRP Developer 10 ml Amber +4 o C K372-100-8 Protein G Coated Plate 1 each ---- -20 o C K372-100-11 BioVision rev. 10/07
|
|
Application Notes
|
Note: 5 μ l is needed for each assay): Mix 1 volume of Acetylating Reagent A (Violet cap) with two volumes of Acetylating Reagent B (Amber cap) in a microtube. Prepare just enough for the experiment. Use within 1 hour. 8. Add 5 μ l of the Acetylating Reagent Mix directly into each test solution (both standard and sample), IMMEDIATELY vertex 2-3 seconds following each addition, and incubate at room temperature for 10 min. 9. Add 845 μ l 1X Assay Buffer into each tube, mix well. Use for below quantification. The acetylation step improves the assy sensitivity significantly and avoid the interferences of many components in unpurified samples. Component K372-100 Color Code Storage Part 100 assays Cap Color Temperature Number 10X cGMP Assay Buffer 25 ml WM +4 o C K372-100-1 Standard cGMP (10 nmol) 1 vial Yellow -20 o C K372-100-2 Neutralizing Buffer 7.5 ml NM +4 o C K372-100-3 Acetylating Reagent A 0.75 ml Violet +4 o C K372-100-4 Acetylating Reagent B 1.5 ml Brown +4 o C K372-100-5 Anti-cGMP pAb/BSA 1 vial Red -20 o C K372-100-6 cGMP-HRP/BSA 1 vial Green -20 o C K372-100-7 HRP Developer 10 ml Amber +4 o C K372-100-8 Protein G Coated Plate 1 each ---- -20 o C K372-100-11 BioVision rev. 10/07 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 cGMP: 1. Add 50 μ l of the acetylated Standard cGMP and test samples from Step 9 above to each well of the Protein G coated 96-well plate. We suggest duplicate assays for each sample and standard. 2. Add 10 μ l of the reconstituted cGMP antibody per well to the standard cGMP and sample wells except the well with 0 pmol cGMP. (Do not add cGMP antibody into the well with 0 pmol cGMP, instead add 10 μ l of 1X Assay Buffer for background reading). Incubate for 1 hour at room temperature with gentle agitation. Using a repeating pipette is recommended for minimizing pippetting errors. 3. Add 10 μ l of cGMP-HRP to each well and incubate for 1 hr at room temperature with gentle agitation. 4. Wash 5 times with 200 μ l 1X Assay Buffer each time 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 sample at OD 450 nm. 8. Subtract OD450 background reading (the well with 0 pmol cGMP) from all samples and standards. Make standard curve. Apply OD readings from samples to the standard curve. Calculate amount of cGMP in samples after correcting the dilution factors. 9. Calculations: C = Sa/Sv pmol/ μ l or nmol/ml or μ M. Where: Sa is the cGMP amount (pmol) from the Standard Curve. Sv is the sample volume ( μ l) added into the assay wells after dilution factor correctionn. IV.
|
