Product Details
| abID | ab65355 |
|---|---|
| Cat # +Size | K371-100 |
| Size | 100 assays |
| Detection Method | Absorbance (450 nm) |
| Species Reactivity | Mammalian |
| Applications | The kit can detect ~0.1-10 pmol/5 μl (or ~ 0.02-2 μM) cAMP samples. |
| Features & Benefits | • Simple procedure • Fast and convenient • The assay is sensitive, stable and high-throughput adaptable • The kit provides a new acetylation procedure to improve detection sensitivity significantly |
| Kit Components | • 10X cAMP Assay Buffer • Standard cAMP (10 nmol) • Neutralizing Buffer • Acetylating Reagent A • Acetylating Reagent B • Rabbit Anti-cAMP pAb • HRP Developer • Protein G Coated Plate |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Adenosine 3’,5’-cyclic monophosphate (cyclic AMP, cAMP) is an important “second messenger” involved in many physiological processes. BioVision’s cAMP Assay Kit provides a direct competitive immunoassay for sensitive and quantitative determination of cAMP level in biological samples. The kit utilizes recombinant Protein G coated 96-well plate to efficiently anchor cAMP polyclonal antibody on to the plate. cAMP-HRP conjugate directly competes with cAMP from sample binding to the cAMP antibody on the plate. After incubation and washing, the amount of cAMP-HRP bound to the plate can easily be determined by reading HRP activity at OD450 nm. The intensity of OD450 nm is inversely proportional to the concentration of cAMP in samples. In addition, the kit provides a new acetylation procedure to improve detection sensitivity significantly. The kit can detect ~0.1-10 pmol/5 μl (or ~ 0.02-2 μM) cAMP samples.
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The tissues/cells need to be homogenized in 0.1M HCl. Would it affect the BCA assay?
Up to 0.1 M HCl (upper limit for HCl in BCA assay) seems to be compatible with BCA assay. Therefore, you can use BCA assay to measure protein amount in your samples.
Guanosine 3,5-cyclic monophosphate (cyclic GMP, cGMP) has been shown to be present at levels typically 10-100 fold lower than cAMP in most tissues. How does this fit with the cAMP Direct Immunoassay Kit to be much more sensitive, 1~100 fmol of cAMP per assay, than the cGMP Direct Immunoassay Kit, 0.1-10 pmol cGMP per assay?
The cAMP antibody affinity is higher than that of cGMP. That is the reason why you need fmol of cAMP while for cGMP you need pmol quantities.
Would the release of hemoglobin from lysed RBC interfere with the read-out?
The hemoglobin will not bind to the antibody in the plate. It will go out with the wash.
How much sample do we need?
10 Million cells or 100 mg of tissue
What can I do if I am not getting cAMP signal from my sample?
A possibility is that esterase degraded the cAMP. So preserve your sample fresh, and use 0.1M HCl lyse your sample immediately. cAMP is stable in 0.1M HCl.
Our customer is using a desiccator connected to vacuum pump. Even 100 ul of the sample did not get dried in nearly 2 hrs. What do you think?
Yes, the vacuum desiccation actually is very slow and would not be my first choice. Most labs generally have a bench-top vacuum centrifuge which I think would be ideal for this purpose, but if not, the oven drying should work as well. Use an oven at ~60°C.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Ana I. Duarte, et al., (2018) Dual Therapy with Liraglutide and Ghrelin Promotes Brain and Peripheral Energy Metabolism in the R6/2 Mouse Model of Huntington’s Disease, Scientific Reports, Jun. 2018, 29895889
Bo, Zhang et al. (2017) G Protein Alpha S Subunit Promotes Cell Proliferation of Renal Cell Carcinoma with Involvement of Protein Kinase A Signaling, DNA Cell Biol. 2017 Mar;36(3):237-242.
Zou, Shenglong et al. (2017) Somatostatin receptor 5 is a prominent regulator of signaling pathways in cells with coexpression of Cannabinoid receptors 1, Neuroscience. 2017 Jan 6;340:218-231.
Sun, Yan-Yan et al. (2016) Surface expression of hippocampal NMDA GluN2B receptors regulated by fear conditioning determines its contribution to memory consolidation in adult rats Sci Rep. 2016 Aug 4;6:30743. doi: 10.1038/srep30743.
Li and Lytton. An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety. J. Biol. Chem., Sep 2014; 289: 25445 - 25459.
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