cAMP Binding Protein Assay for Widespread Use in Cell Signaling Studies

Abstract

cAMP plays a critical role in intracellular signaling pathways that regulate proliferation or differentiation. The cAMP binding protein assay, using a naturally derived cAMP binding protein, is one of the most widely used methods for cAMP determination. The major steps of this binding assay include purification of the binding protein, cAMP extraction from samples, and quantification of the cAMP Most purification methods of the cAMP binding protein were published before 1975, and many of the materials and methods are outdated. Here we describe an updated method of purification of cAMP binding protein from bovine skeletal muscle with the advantages of simplicity, low cost, and high yield The isolation procedures can be completed in two days using commercially available materials and equipment. The cAMP binding properties of the isolated protein can be utilizedfor more than two years. Binding protein isolatedfrom 1 kg bovine muscle is sufficientfor at least 3 x10(4) assay tubes. Furthemore, we describe the techniques of cAMP extraction and quantification that have been used successfully in studying parathyroid hormone signaling as an example of a G protein-linked seven transmembrane domain receptor that signals through the protein kinase A pathway.

The ABC transporter, AbcB3, mediates cAMP export in D. discoideum development

“…This analog binds to cell surface cAMP receptors and induces cAMP production and export, but it is not detected by the cAMP detection method we used (32, 33). We found that stimulation caused a twofold increase in the levels of extracellular cAMP in the wild type but no significant increase in the two abcB3 -RNAi cells (Fig.…”

“…Briefly, samples were neutralized using a 50% saturated solution of KHCO 3 , centrifuged, and the supernatant collected for the assay. 20 μL of tracer (0.4 μCi/mL of 3 H-cAMP), 20 μL of sample or standard, and 40μL of cAMP binding protein prepared from beef muscle (33) were combined and incubated on ice for 1.5 hours. Binding reactions were stopped by addition of 40μL activated charcoal.…”

High Throughput Screening (HTS) in Identification New Ligands and Drugable Targets of G Protein-Coupled Receptors (GPCRs)

“…Studies on activation of both G s -and G i -coupled receptors are focus on cAMP [97]. The difference is that they lead adenylate cyclase activation (G s ) or inhibition (G i ), resulting in increased or decreased intracellular cAMP levels, respectively.…”

Pulsatile release of parathyroid hormone from an implantable delivery system

“…The in vitro bioactivity of released PTH was determined by adenylate cyclase stimulation assay and cAMP binding protein assay as previously described [38,39]. Briefly, rat osteosarcoma cells (ROS 17/2.8) were treated with PTH (1-34) of known concentrations or with eluent from the PTH delivery device for designated times in calcium-and magnesium-free hanks' balanced salt solution (Invitrogen, Carlsbad, CA) containing 0.1% BSA and 1 mM isobutylmethylxanthine (IBMX).…”