cAMP ELISA Kit (Cyclic Adenosine Monophosphate)

Details for Product cAMP ELISA Kit No. ABIN2345107, Supplier: Log in to see
Antigen
  • CRAMP
  • CAP-18
  • CAP18
  • FALL-39
  • FALL39
  • LL37
  • CAP11
  • Cnlp
  • Cramp
  • MCLP
  • CATHL7
  • cathelicidin antimicrobial peptide
  • Camp
  • CAMP
Alternatives
Guinea Pig cAMP ELISA Kit
Reactivity
Chemical
Alternatives
Kits with alternative reactivity to:
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Method Type
Competition ELISA
Application
ELISA
Options
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Purpose An anti-Rabbit IgG polyclonal coating antibody is adsorbed onto a microtiter plate. Cyclic AMP present in the sample or standard competes with Peroxidase cAMP Tracer for plate binding, in the presence of Rabbit Anti-cAMP Polyclonal Antibody. Following incubation and wash steps, any Peroxidase cAMP Tracer bound to the plate is detected with addition of Chemiluminescent Reagent. The light product formed is inversely proportional to the amount of cAMP present in the sample. This reaction is then measured in a plate luminometer. A standard curve is prepared from cAMP Standard and sample concentration is then determined. 2
Sample Type Plasma, Urine
Analytical Method Quantitative
Detection Method Chemiluminescent
Sensitivity 1 pmol/mL
Characteristics cAMP ELISA Kit is a competitive enzyme immunoassay designed to measure cAMP in cell culture supernatants, plasma, serum, saliva, urine, and cell lysates. The kit selectively measures cAMP levels without any significant cross reactivities to other nucleotides or cyclic nucleotides. Samples containing low cAMP levels may be acetylated (reagents provided) for increased sensitivity. Under non-acetylated conditions, the kit has a detection range of 1 to 1000 pmol/mL cAMP, however, under acetylated conditions, the sensitivity is enhanced (approx 100X) to a detection range of 10-2500 fmol/mL.
Components
  1. Goat Anti-Rabbit Antibody Coated Plate : One strip well 96-well plate.
  2. cAMP Standard : One 100 μL vial provided at 10 mM.
  3. Rabbit Anti-cAMP Polyclonal Antibody : One 15 μL vial.
  4. Peroxidase cAMP Tracer Conjugate : One 30 μL vial.
  5. Assay Diluent : One 25 mL bottle.
  6. Lysis Buffer : One 50 mL bottle.
  7. 10X Wash Buffer : One 50 mL bottle.
  8. Triethylamine : One 2 mL amber bottle.
  9. Acetic Anhydride : One 1 mL amber bottle.
  10. Chemiluminescent Reagent A : One 6 mL amber bottle.
  11. Chemiluminescent Reagent B (Part. No. 250103): One 6 mL bottle.
Material not included
  1. Orbital plate shaker
  2. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
  3. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
  4. Multichannel micropipette reservoir
  5. 96-well Plate Luminometer
  6. Glass or polypropylene tubes for acetylated samples and standards
Alternative Name cAMP (cAMP ELISA Kit Abstract)
Background Adenosine 3',5'-cyclic monophosphate (cAMP) is a ubiquitous second messenger involved in various cellular activities in many cell and tissue types. It is converted from adenosine triphosphate (ATP) via adenylyl cyclases (AC), and is inactivated by hydrolysis to 5´-AMP by the actions of phosphodiesterases. cAMP may affect cellular function through several different mechanisms including the activation of cAMP-dependent Protein Kinase (PKA), Guanine Nucleotide Exchange Factors (GEFs), and Cyclic Nucleotide-gated (CNG) channels. PKA is a heterotetramer consisting of 2 regulatory (R) subunits and 2 catalytic (C) subunits. Two cAMP molecules bind cooperatively to 2 sites on each R subunit, releasing the active C subunit monomers to phosphorylate a range of downstream substrates. GEFs facilitate the exchange of GDP for GTP and, therefore, promote the activity of G proteins. Exchange Protein Activated by cAMP (Epac) 1 and 2 are GEFs activated upon binding to cAMP. Epac 1 and 2 have been implicated in regulating the activity of the small GTPase Rap-1 (26, 27). CNG channels are cation channels activated by cGMP and/or cAMP. These channels regulate membrane potential, and due to their Ca2+ permeability, can alter the levels of intracellular Ca2+.
Research Area Signaling
Pathways Cellular Response to Molecule of Bacterial Origin
Comment

  • Sensitivity as low as 1 pmol/mL
  • Suitable for use with cell and tissue lysates, urine, plasma, or culture medium
  • Convenient strip-well plate format

Plate Uncoated
Reagent Preparation
  • 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
  • Rabbit Anti-cAMP Polyclonal Antibody: Immediately before use dilute the Rabbit Anti-cAMP Antibody 1:500 with Assay Diluent. Do not store diluted solutions. 3
  • Peroxidase cAMP Tracer Conjugate: Immediately before use dilute the Peroxidase cAMP Tracer Conjugate 1:100 with Assay Diluent. Do not store diluted solutions.
  • Chemiluminescent Reagent: Immediately before use, mix equal volumes of Chemiluminescent Reagent A with Chemiluminescent Reagent B. Do not store diluted solutions.
  • Acetylation Reagent: Preparation of the Acetylation Reagent should be done in glass tubes and in a fume hood. The Acetylation Reagent is made by mixing Acetic Anhydride with Triethylamine at a 1:2 ratio (example: 0.5 mL Acetic Anhydride + 1 mL Triethylamine). Use the reagent within 60 minutes of preparation. Caution: The components of this reagent are known to be caustic, corrosive, flammable, and lachrymators. Use appropriate protection when handling. Preparation of cAMP Standards (Non-Acetylated Version) 1. Thaw the cAMP Standard at room temperature and mix thoroughly by pipetting (cAMP can precipitate when frozen but will redissolve when mixed well). Freshly prepare a dilution series of cAMP Standard in the concentration range of 100 μM - 100 pM by diluting the cAMP Standard in Lysis Buffer (Table 1). Lysis Buffer cAMP Standard Tubes cAMP Standard (μL) (μL) Concentration 1 10 990 100 μM 2 20 of Tube #1 180 10 μM 3 20 of Tube #2 180 1 μM 4 20 of Tube #3 180 100 nM 5 20 of Tube #4 180 10 nM 6 20 of Tube #5 180 1 nM 7 20 of Tube #6 180 100 pM 8 0 180 0 Table 1.
Assay Procedure
  1. Prepare and mix all reagents thoroughly before use.
  2. Each cAMP sample, cAMP Standard, and blank should be assayed in duplicate. Note: cAMP samples must be compared with corresponding standards (i.e. acetylated samples compared with acetylated standards, non-acetylated samples with non-acetylated standards).
  3. Add 50 μL of cAMP sample or standard (acetylated or non-acetylated) to the Goat Anti-Rabbit Antibody Coated Plate.
  4. Add 25 μL of diluted Peroxidase cAMP Tracer Conjugate (see Preparation of Reagents Section) to each tested well.
  5. Add 50 μL of diluted Rabbit Anti-cAMP Polyclonal Antibody (see Preparation of Reagents Section) to each tested well.
  6. Cover with a Plate Cover and incubate at room temperature for 30 minutes with shaking.
  7. Remove Plate Cover and empty wells. Wash microwell strips 5 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
  8. Add 100 μL of Chemiluminescent Reagent (see Preparation of Reagents Section) to each well, including the blank wells. Incubate at room temperature for 5 minutes on an orbital shaker.
  9. Read the luminescence of each microwell on a plate luminometer. 6
Restrictions For Research Use only
Storage 4 °C/-20 °C
Storage Comment Store kit components at 4°C. For longer term use, store the Rabbit Anti-cAMP Polyclonal Antibody at -20°C.
Product cited in: Israeli, Rotem, Elia, Bar-Haim, Cohen, Chitlaru: "A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity." in: Toxins, Vol. 8, Issue 8, 2016 (PubMed).

Liu, Gritz, Parent: "PKC?II acts downstream of chemoattractant receptors and mTORC2 to regulate cAMP production and myosin II activity in neutrophils." in: Molecular biology of the cell, Vol. 25, Issue 9, pp. 1446-57, 2014 (PubMed).

Liu, Aerbajinai, Ahmed, Rodgers, Angers, Parent: "Radil controls neutrophil adhesion and motility through ?2-integrin activation." in: Molecular biology of the cell, Vol. 23, Issue 24, pp. 4751-65, 2012 (PubMed).

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