A Simple and Inexpensive Protein Binding Assay for Cyclic AMP in Biological Materials

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Abstract
We have developed a simple and inexpensive method for largecapacity cAMP determination in biological materials. The assay is based on competitive binding of 3H-cAMP to proteins isolated from rabbit skeletal muscle. Bovine serum albumin is added to the incubate to reduce non-specific interference. Separation of free and bound radioactivity is performed by (NH4)2SO4 precipitation allowing determination of either free or bound fraction. In the latter case, all procedures are performed in the same tube, to which is finally added 1.2 ml of scintillation fluid for counting. By this one-tube-procedure a high precision is obtained and the expense per sample is reduced to approximately one fourth. The method has been applied to rat tissue extracts and urine with satisfactory sensitivity, precision and accuracy.