Overview

  • Product name

    cAMP Assay Kit (Competitive ELISA)
    See all cAMP kits
  • Detection method

    Colorimetric
  • Sample type

    Urine, Serum, Other biological fluids, Tissue Extracts, Cell Lysate, Cell culture media
  • Assay type

    Quantitative
  • Sensitivity

    > 0.02 µM
  • Range

    0.02 µM - 2 µM
  • Assay time

    3h 30m
  • Species reactivity

    Reacts with: Mammals, Other species
  • Product overview

    cAMP Assay Kit ab65355 uses a sensitive colorimetric competitive ELISA method to quantify cAMP (cyclic AMP) levels in samples from mammals and other species. It has a sensitivity of 0.02 µM.


    The cAMP assay protocol uses a 96-well plate supplied coated with Protein G. The Protein G binds an anti-cAMP antibody which is added to the plate.


    HRP-labeled cAMP, and free cAMP within a sample, are then added to each well of the plate. The cAMP assay is based on the competition between the HRP-labeled cAMP and the free cAMP for the fixed number of cAMP antibody binding sites on the plate.


    The more free cAMP is present, the less HRP-labeled cAMP is bound. After a washing step, the amount of bound HRP-labeled cAMP is detected using a colorimetric HRP substrate measured by absorbance at OD 450 nm. The level of signal is compared to a standard curve for free cAMP, which is run at the same time as the samples.


    This kit uses an acetylation step to achieve the maximum sensitivity.


    cAMP assay protocol summary:
    - add prepared samples and standards to tubes
    - add neutralizing buffer
    - add acetylating reagent mix, vortex and incubate for 10 min at room temp
    - add assay buffer
    - transfer to protein-G 96-well plate
    - add cAMP antibody and incubate for 1 hr at room temp
    - add cAMP-HRP and incubate for 1 hr at room temp
    - wash with assay buffer
    - add HRP developer and incubate for 1 hr at room temp
    - stop reaction with HCl
    - analyze with microplate reader

  • Notes

    This kit was previously called cAMP Assay Kit - Direct Immunoassay.

    If you are using fluorometric detection, we recommend cAMP Assay Kit (Fluorometric) (ab138880).

     

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Storage buffer

    Preservative: None
  • Components Identifier 100 tests
    10X cAMP Assay Buffer WM 1 x 25ml
    Acetylating Reagent A Violet 1 x 750µl
    Acetylating Reagent B Black 1 x 1.5ml
    cAMP-HRP Green 1 vial
    HRP Developer Amber 1 x 10ml
    Neutralizing Buffer NM 1 x 7.5ml
    Protein G Coated Plate 1 unit
    Rabbit Anti-cAMP polyclonal Antibody Red 1 vial
    Standard cAMP (10 nmol) Yellow 1 x 10µl
  • Research areas

  • Relevance

    Adenosine 3’,5’-cyclic monophosphate (cyclic AMP, cAMP) is an important “second messenger” involved in many physiological processes. It plays a key role as an intracellular second messenger for transduction events that follow a number of extracellular signals.
  • Alternative names

    • Adenosine 3' 5' cyclic monophosphate
    • Adenosine 3’ 5’ cyclic monophosphate
    • Cyclic adenosine monophosphate
    • Cyclic AMP

Images

  • Concentration of cAMP in response to in vitro haemocyte stimulation with LPS and β-AR (B) agonist/antagonist at 24h. Data presented as mean ± S.D. (N = 6) of cAMP concentration expressed as pmol µL-1. The significant differences among the control and treatedgroups were subjected to one-way ANOVA, followed by Student-Newman-Keuls multiple range test, which revealed two groups (a to b). The concentration of cAMP with the same letter in common do not differ at the P = 0.05 level of significance.

  • Measurement of cAMP in Jurkat and HepG2 cells with ab65355. Assay performed using the kit protocol.

  • Standard curve (colorimetric): mean of duplicates (+/-SD)

Protocols

References

This product has been referenced in:

  • Chen S  et al. Targeting MC1R depalmitoylation to prevent melanomagenesis in redheads. Nat Commun 10:877 (2019). Read more (PubMed: 30787281) »
  • Chen WC  et al. Xenotropic and polytropic retrovirus receptor 1 (XPR1) promotes progression of tongue squamous cell carcinoma (TSCC) via activation of NF-?B signaling. J Exp Clin Cancer Res 38:167 (2019). Read more (PubMed: 30995931) »
See all 25 Publications for this product

Customer reviews and Q&As

1-10 of 17 Abreviews or Q&A

Answer

As this kit will detect a cyclic nucleotide and not a protein, it does not have a species restriction and should be fine for the use with bacteria.

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Question
Answer

We recommend starting with 10 Million cells or 100 mg of tissue.

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Question
Answer

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.

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Answer

You are correct. There is no need to add a blocking step to this protocol. Further you do not need to wash the plates prior to adding the antibody or cAMP-HRP.

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The kit was easier to use and worked as expected. Directions for sample prep were followed and we had measurable values of cAMP on this assay. The protocols are fairly simple but required thorough review to understand certain steps. Our sample pool in serial dilution performed similar to the standards, however need to be diluted less than the kit recommends for our sample type. We will continue using this assay in the future.

Abcam user community

Verified customer

Submitted Sep 21 2017

Answer

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 ˜60oC.

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Answer

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.

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Question
Answer

I am pleased to provide the suggested protocol for preparing cell lysate samples for this assay:

Cell Samples:

Aspirate medium.
Add 1 ml of 0.1M HCl for every 35 cm2 of surface area.
Incubate at room temperature for 20 min.
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.

This information can be found in the protocol provided with the online datasheet, page 7.

https://www.abcam.com/ps/products/65/ab65355/documents/ab65355%20cAMP%20Direct%20Immunoassay%20Kit%20(Website).pdf

(This can be found by clicking on the protocols tab on the online datasheet, and then selecting the protocols booklet link)

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Question
Answer

The hemoglobin will not bind to the antibody in the plate. It will go out with the wash.

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Answer

In regards to your question as to whether ourcAMP Direct Immunoassay Kit, ab65355, will work for your need, yes this assay can work beautifully for such a purpose. Please use all appropriate background controls.

This product is covered by our Abpromise guarantee. We provide scientific support, replacement or refund should this product not perform as indicated on the datasheet. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise

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1-10 of 17 Abreviews or Q&A

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