cAMP binding protein assay for widespread use in cell signaling studies.
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purification methods of the cAMP binding protein were published before 1975, and many of the materials and methods are outdated.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.
Chen
Hen-Li
HL
University of Michigan, Ann Arbor 48109-1078, USA.
McCauley
Laurie K
LK
D'Silva
Nisha J
NJ
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose / Q000379:methods*; D002373:Cyclic AMP Receptor Protein / Q000302:isolation & purification* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D010006:Osteoblasts / Q000145:classification / Q000187:drug effects / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000494:pharmacology; D014677:Pharmaceutical Vehicles / Q000494:pharmacology; D012016:Reference Values; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000502:physiology*; D013045:Species Specificity
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D014677:Pharmaceutical Vehicles; C432808:parathyroid hormone-related protein (1-139); D000262:Adenylyl Cyclases