Standard Curve (Sample)
cAMP ELISA Kit
Elite cAMP ELISA Assay Kit (Fluorescence)
Synonyms
cAMP; N/A; Elite cAMP ELISA Assay Kit (Fluorescence); cAMP ELISA Assay Kit (Fluorescence); cAMP elisa kit
Features
• Wide reading windows
• High sensitivity
• Sensitivity (IC50): 1.8 nM (1.8 pmol/mL)
• High sensitivity
• Sensitivity (IC50): 1.8 nM (1.8 pmol/mL)
Kit Components
• Component A: cAMP Standard 33 ug (store at -20 degree C)
• Component B: Assay Buffer 20 mL
• Component C: HRP-cAMP Conjugate 1 vial (store at -20 degree C)
• Component D: 10x Wash Buffer 10 mL
• Component E: Cell Lysis Buffer 10 mL
• Component F: 3% H2O2 50 uL
• Component G: EliteTM Red 1 vial (store at -20 degree C)
• Component H: Anti-cAMP Antibody Pre-Coated 96-Well Plate 1 plate
• Component I: Substrate Buffer 10 mL
• Component B: Assay Buffer 20 mL
• Component C: HRP-cAMP Conjugate 1 vial (store at -20 degree C)
• Component D: 10x Wash Buffer 10 mL
• Component E: Cell Lysis Buffer 10 mL
• Component F: 3% H2O2 50 uL
• Component G: EliteTM Red 1 vial (store at -20 degree C)
• Component H: Anti-cAMP Antibody Pre-Coated 96-Well Plate 1 plate
• Component I: Substrate Buffer 10 mL
Assay Protocol for 96-Well Plate:
1. Prepare cells or samples:
1.1 Cell Samples:
For adherent cells: Plate cells overnight in growth medium at 30,000 -100,000 cells/well for a 96-well plate.
For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellets in culture medium at 100,000-300,000 cells/well for a 96-well poly-D lysine plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiment.
Treat cells as desired: The following is an example for Hela cells treated with Forskolin to induce cAMP in a 96-well plate format.
* Aspirate off cell growth medium, add 100 uL/well of 0.75 mM IBMX in Hanks and 20 mM Hepes buffer (HHBS). Incubate at room temperature for 10 minutes.
* Add 50 uL/well of 150 uM Forskolin in HHBS, incubate in a 5% CO2, 37 degree C incubator for 15 minutes.
* Aspirate off cell solution after the incubation. And then add 100 uL/well of Cell Lysis Buffer (Component E), incubate at room temperature for another 10 minutes.
* This cell lysate can be assayed directly or diluted in cell lysis buffer.
Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density. Cells may be seeded the day before or on the day of the experiment depending upon the cell type and/or the effect of the test compounds.
1.2 Tissue Samples: It is important to rapidly freeze tissues after collection (e.g., using liquid nitrogen) due to quick metabolism of cyclic nucleotides in tissue. Weigh the frozen tissue and add 10-20 uL/mg of cell lysis buffer. Homogenize the sample on ice. Spin at top speed for 5 minutes and collect the supernatant. The supernatant may be assayed directly.
1.3 Urine, Plasma and Culture Medium Samples: Urine and plasma may be tested directly with 1:200 to 1:1000 dilutions in Lysis Buffer. Culture medium can also be tested with 1:10 to 1:200 dilutions in Lysis Buffer.
Note: RPMI medium may contain > 350 fmol/uL cAMP.
2. Prepare cAMP assay solutions:.
2.1 cAMP Stock Solution (100 uM): add 1 mL of Assay Buffer (Component B) to the vial of cAMP Standard (Component A).
2.2 Prepare serial dilution of cAMP in Assay Buffer (Component B) to have 10,000, 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0.003 nM cAMP diluted solutions. Store on ice or 4 degree C.
Note: The unused 100 uM cAMP stock solution should be aliquoted and stored at -20 degree C.
2.3 HRP-cAMP Conjugate Stock Solution (50x): add 55 uL of Assay Buffer (Component B) into the vial of HRP-cAMP Conjugate (Component C).Note: The unused 50x HRP-cAMP conjugate stock solution should be aliquoted and stored at -20 degree C.
2.4 Prepare 1X HRP-cAMP conjugate working solution by adding 50 uL HRP-cAMP Conjugate Stock Solution (50x) into 2.45 mL of Assay Buffer (Component B). Store on ice or 4 degree C.
2.5 Prepare 1X washing buffer by adding 1 mL of 10X Wash Buffer (Component D) to 9 ml distilled water.
2.6 EliteTM Red Stock Solution (200x): add 50 uL of DMSO to the vial of EliteTM Red (Component G).
3. Run cAMP assay:
3.1. All the assay wells will be prepared in the following orders: A) cAMP standards, control, or tests samples; B) HRP-cAMP conjugate.
3.2. Add 75 uL/well of the cAMP diluted solution (from Step 2.2) and test samples into each well of the anti-cAMP Ab pre-coated 96-well plate (Component H). It is recommended to duplicate the assays for each standard and test sample. Incubate at room temperature for 5 to 10 minutes.
3.3. Add 25 uL/well of 1X HRP-cAMP conjugate working solution (from Step 2.4). Incubate at room temperature for 2 hours by placing the plate on shaker.
3.4. Aspirate plate contents, and wash 4 times with 200 uL/well of 1X wash buffer (from Step 2.5).
3.5. Prepare Elite™ Red working solution by adding 50 uL of Elite™ Red stock solution (200x) and 11.5 ul of 3% H2O2 (Component F) into 10 ml of Substrate Buffer (Component I).
Note: The Elite™ Red working solution is not stable, use it promptly.
3.6. Add 100 uL/well of Elite™ Red working solution (from Step 3.5) into each well, and incubate at room temperature for 10 minutes to 2 hours, protected from light.
3.7. Monitor the fluorescence increase at Ex/Em = 540/590 nm (cutoff 570 nm) by using a fluorescence plate reader (top read mode). For best result, get the reading within 1-2 hours after incubation.
1.1 Cell Samples:
For adherent cells: Plate cells overnight in growth medium at 30,000 -100,000 cells/well for a 96-well plate.
For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellets in culture medium at 100,000-300,000 cells/well for a 96-well poly-D lysine plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiment.
Treat cells as desired: The following is an example for Hela cells treated with Forskolin to induce cAMP in a 96-well plate format.
* Aspirate off cell growth medium, add 100 uL/well of 0.75 mM IBMX in Hanks and 20 mM Hepes buffer (HHBS). Incubate at room temperature for 10 minutes.
* Add 50 uL/well of 150 uM Forskolin in HHBS, incubate in a 5% CO2, 37 degree C incubator for 15 minutes.
* Aspirate off cell solution after the incubation. And then add 100 uL/well of Cell Lysis Buffer (Component E), incubate at room temperature for another 10 minutes.
* This cell lysate can be assayed directly or diluted in cell lysis buffer.
Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density. Cells may be seeded the day before or on the day of the experiment depending upon the cell type and/or the effect of the test compounds.
1.2 Tissue Samples: It is important to rapidly freeze tissues after collection (e.g., using liquid nitrogen) due to quick metabolism of cyclic nucleotides in tissue. Weigh the frozen tissue and add 10-20 uL/mg of cell lysis buffer. Homogenize the sample on ice. Spin at top speed for 5 minutes and collect the supernatant. The supernatant may be assayed directly.
1.3 Urine, Plasma and Culture Medium Samples: Urine and plasma may be tested directly with 1:200 to 1:1000 dilutions in Lysis Buffer. Culture medium can also be tested with 1:10 to 1:200 dilutions in Lysis Buffer.
Note: RPMI medium may contain > 350 fmol/uL cAMP.
2. Prepare cAMP assay solutions:.
2.1 cAMP Stock Solution (100 uM): add 1 mL of Assay Buffer (Component B) to the vial of cAMP Standard (Component A).
2.2 Prepare serial dilution of cAMP in Assay Buffer (Component B) to have 10,000, 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0.003 nM cAMP diluted solutions. Store on ice or 4 degree C.
Note: The unused 100 uM cAMP stock solution should be aliquoted and stored at -20 degree C.
2.3 HRP-cAMP Conjugate Stock Solution (50x): add 55 uL of Assay Buffer (Component B) into the vial of HRP-cAMP Conjugate (Component C).Note: The unused 50x HRP-cAMP conjugate stock solution should be aliquoted and stored at -20 degree C.
2.4 Prepare 1X HRP-cAMP conjugate working solution by adding 50 uL HRP-cAMP Conjugate Stock Solution (50x) into 2.45 mL of Assay Buffer (Component B). Store on ice or 4 degree C.
2.5 Prepare 1X washing buffer by adding 1 mL of 10X Wash Buffer (Component D) to 9 ml distilled water.
2.6 EliteTM Red Stock Solution (200x): add 50 uL of DMSO to the vial of EliteTM Red (Component G).
3. Run cAMP assay:
3.1. All the assay wells will be prepared in the following orders: A) cAMP standards, control, or tests samples; B) HRP-cAMP conjugate.
3.2. Add 75 uL/well of the cAMP diluted solution (from Step 2.2) and test samples into each well of the anti-cAMP Ab pre-coated 96-well plate (Component H). It is recommended to duplicate the assays for each standard and test sample. Incubate at room temperature for 5 to 10 minutes.
3.3. Add 25 uL/well of 1X HRP-cAMP conjugate working solution (from Step 2.4). Incubate at room temperature for 2 hours by placing the plate on shaker.
3.4. Aspirate plate contents, and wash 4 times with 200 uL/well of 1X wash buffer (from Step 2.5).
3.5. Prepare Elite™ Red working solution by adding 50 uL of Elite™ Red stock solution (200x) and 11.5 ul of 3% H2O2 (Component F) into 10 ml of Substrate Buffer (Component I).
Note: The Elite™ Red working solution is not stable, use it promptly.
3.6. Add 100 uL/well of Elite™ Red working solution (from Step 3.5) into each well, and incubate at room temperature for 10 minutes to 2 hours, protected from light.
3.7. Monitor the fluorescence increase at Ex/Em = 540/590 nm (cutoff 570 nm) by using a fluorescence plate reader (top read mode). For best result, get the reading within 1-2 hours after incubation.
Preparation and Storage
Keep Component A, C, G at -20 degree C and all other Components at 4 degree C; keep Component G in the dark.
Standard Curve (Sample)
Standard Curve (Sample)
Related Product Information for cAMP elisa kit
Principle of the Assay: Adenosine 3', 5' cyclic monophosphate (cAMP) is an important second messenger in intracellular signal transduction. Monitoring cAMP levels is one of the most common ways to screen for agonists and antagonists of GPCRs. Elite™ cAMP ELISA Assay Kit is based on the competition between HRP-labeled cAMP and free cAMP for a fixed number of cAMP antibody binding sites. HRP-cAMP is displaced from the HRP-cAMP/anti-cAMP antibody complex by unlabeled free cAMP. In the absence of cAMP, HRP-cAMP conjugate is bound to anti-cAMP antibody exclusively. However, the unlabeled free cAMP in the test samples competes for anti-cAMP antibody with the HRP-cAMP antibody conjugate, therefore inhibits the binding of HRP-cAMP to anti-cAMP antibody. Elite™ cAMP ELISA Assay Kit provides the sensitive method for detecting adenylate cyclase activity in biochemical or cell-based assay system. Compared to other ELISA cAMP assay kits, our kit eliminates the tedious acetylation step. The kit uses Elite™ Red as a fluorimetric substrate to quantify the HRP activity. The assay can be performed in a convenient 96-well microtiter-plate format and easily adapted to automation. The fluorescent product formed is proportional to the activity of HRP-cAMP conjugate.
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Product Notes
The cAMP (Catalog #AAA62153) is an ELISA Kit and is intended for research purposes only. The product is available for immediate purchase. It is sometimes possible for the material contained within the vial of "cAMP, ELISA Kit" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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