Adenylate cyclase assay with [α-32P]ATP as substrate: A simple modification for lowering blanks
✍ Scribed by Raymond Counis; Sylvie Mongongu
- Publisher
- Elsevier Science
- Year
- 1978
- Tongue
- English
- Weight
- 414 KB
- Volume
- 84
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
In the assay of adenylate cyclase using [&*P]ATP as the substrate and alumina chromatography as the separating procedure for labeled nucleotides, blank levels are dependent on the quality of the labeled ATP and also on that of the alumina. In order to lower the blanks by eliminating the radioactive material contaminating the commercial [aJZP]ATP preparations, the following treatment is proposed: The reaction mixture resulting from the incubation is heated for 4 min at 95°C in 0.165 N HCI, then it is chromatographed on a selected alumina (Woelm) column. In the conditions used, cyclic AMP was unaffected, while blank values were low. The detection limit of [32P]cyclic AMP was thus higher and the precision of enzyme activity determination was improved, while the advantages of one-step chromatography were retained.