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Signal Transduction Second Messenger Nucleotide Messenger cAMP
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cAMP Assay Kit (Competitive ELISA) (ab65355)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (2)Q&A (16)References (83)

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Functional Studies - cAMP Assay Kit (Competitive ELISA) (ab65355)
  • Functional Studies - cAMP Direct Immunoassay Kit (ab65355)
  • Functional Studies - cAMP Direct Immunoassay Kit (ab65355)

Key features and details

  • Assay type: Quantitative
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 3 hr 30 min
  • Sample type: Cell culture media, Cell Lysate, Other biological fluids, Serum, Tissue Extracts, Urine
  • Sensitivity: 0.002 µM

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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

    > 0.002 µM
  • Range

    0.002 µM - 0.2 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 0.002 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 product is manufactured by BioVision, an Abcam company and was previously called K371 cAMP Direct Immunoassay Kit (Colorimetric). K371-100 is the same size as the 100 test size of ab65355.

    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.
  • 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

    • Signal Transduction
    • Second Messenger
    • Nucleotide Messenger
    • cAMP
    • Kits/ Lysates/ Other
    • Kits
    • Cell Signaling Kits
    • cAMP/cGMP assay kits
  • 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

Associated products

  • Assay kits

    • cAMP Assay Kit (Competitive ELISA, Fluorometric) (ab138880)
    • cAMP Assay Kit (Competitive ELISA) (ab234585)
  • Related Products

    • 8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP), Cell permeable cAMP analog. (ab120424)
    • Adenosine 3prime,5prime-cyclic monophosphate (cAMP), Second messenger. PKA activator. (ab120493)
    • 8-Br-cAMP (8-Bromo-cAMP), PKA activator (ab141448)
    • RHC-80267, diacylglycerol lipase inhibitor (ab141758)
    • 10kD Spin Column (ab93349)

Images

  • Functional Studies - cAMP Assay Kit (Competitive ELISA) (ab65355)
    Functional Studies - cAMP Assay Kit (Competitive ELISA) (ab65355)Jiang, Qiufen et al., Scientific reports vol. 4 6963., Fig 4, doi:10.1038/srep06963

    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.

  • Functional Studies - cAMP Direct Immunoassay Kit (ab65355)
    Functional Studies - cAMP Direct Immunoassay Kit (ab65355)

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

  • Functional Studies - cAMP Direct Immunoassay Kit (ab65355)
    Functional Studies - cAMP Direct Immunoassay Kit (ab65355)

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

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (83)

Publishing research using ab65355? Please let us know so that we can cite the reference in this datasheet.

ab65355 has been referenced in 83 publications.

  • Mohammed I  et al. A Novel Homozygous MC2R Variant Leading to Type-1 Familial Glucocorticoid Deficiency. J Endocr Soc 6:bvac058 (2022). PubMed: 35506146
  • Ruiz de Martín Esteban S  et al. Cannabinoid CB2 Receptors Modulate Microglia Function and Amyloid Dynamics in a Mouse Model of Alzheimer's Disease. Front Pharmacol 13:841766 (2022). PubMed: 35645832
  • Ibrahim SSA  et al. Anti-neoplastic action of Cimetidine/Vitamin C on histamine and the PI3K/AKT/mTOR pathway in Ehrlich breast cancer. Sci Rep 12:11514 (2022). PubMed: 35798765
  • Martínez-Rojas B  et al. NPC transplantation rescues sci-driven cAMP/EPAC2 alterations, leading to neuroprotection and microglial modulation. Cell Mol Life Sci 79:455 (2022). PubMed: 35904607
  • Chaffin AT  et al. FGF21 controls hepatic lipid metabolism via sex-dependent interorgan crosstalk. JCI Insight 7:N/A (2022). PubMed: 35998055
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 18 Abreviews or Q&A

Acetylating Reagent B Unable to Retrieve!

Below average Poor 1/5 (Ease of Use)
Abreviews
Abreviews
The "Acetylating Reagent B" was placed in a glass vial where I do not even understand how you were able to place into. However, after several attempts to shake the tube out of the glass vial, I ended up having to shatter the glass vial to retrieve the tube.

Next time, for the convenience of customers, perhaps you should consider preparing a glass vial big enough to easily take the tube out. Simply ridiculous.

Abcam reply

Thank you for your feedback. We are really sorry to hear that you had some trouble getting the Acetylating Reagent B from the glass vial it has been stored in. The Acetylating Reagent B is very volatile hence, stored in two containers. We are currently looking into alternative vial types for this reagent and will fix this issue as soon as possible. I sincerely apologise for the inconvenience caused.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Dec 21 2022

cAMP in dog follicles and in culture medium collected from dog follicle culture

Excellent Good 4/5 (Ease of Use)
Abreviews
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Sep 21 2017

Question

I want to mesure the intracellular level of c-AMP in bacteria and I want to know if your cAMP enzyme immunoassay kit can be used for this assay?

Read More

Abcam community

Verified customer

Asked on Feb 19 2014

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.

Read More

Abcam Scientific Support

Answered on Feb 19 2014

Question

How much sample do we need?

Read More

Abcam community

Verified customer

Asked on May 18 2012

Answer

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

Read More

Abcam Scientific Support

Answered on May 18 2012

Question

What can I do if I am not getting cAMP signal from my sample?

Read More

Abcam community

Verified customer

Asked on May 08 2012

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.

Read More

Abcam Scientific Support

Answered on May 08 2012

Question

Protocol does not metion about blocking, so it means this direct ELISA kit doesn't need this step? Do I need wash the plate before add antibody and cAMP-HRP?

Read More

Abcam community

Verified customer

Asked on Apr 19 2012

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.

Read More

Abcam Scientific Support

Answered on Apr 19 2012

Question

In the protocol provided (3. cAMP assay), it states that If cAMP in your samples are very low, the acetylation reagents can be dried after step 3-e, without dilution step 3-d to minimize the volume increase. May i know how to do the drying before reconstitution in assay buffer?

Read More

Abcam community

Verified customer

Asked on Nov 20 2015

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.

Read More

Sam Washer

Abcam Scientific Support

Answered on Nov 20 2015

Question

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?

Read More

Abcam community

Verified customer

Asked on Feb 06 2014

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.

Read More

Padamjeet Singh

Abcam Scientific Support

Answered on Feb 06 2014

Question

Could you please advise what reagents should be used to lyse the cultured cells?

Read More

Abcam community

Verified customer

Asked on Oct 02 2012

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.

Read More

Abcam Scientific Support

Answered on Oct 02 2012

Question

Would the release of hemoglobin from lysed RBC interfere with the read-out?

Read More

Abcam community

Verified customer

Asked on Aug 21 2012

Answer

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

Read More

Abcam Scientific Support

Answered on Aug 21 2012

1-10 of 18 Abreviews or Q&A

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