Rp-2′-AHC-cAMPS-Agarose

Chromatography : After equilibration with about 10 column volumes of starting buffer the affinity column is loaded with the protein solution at 50 – 100 μl/min. In order to elute other nucleotide-dependent proteins unspecifically bound, the column is washed, e.g. with 1mM 5′- AMP. Elution of the target protein is performed by a cyclic nucleotide gradient. For elution of phosphodiesterases it could be advisable to use the highly hydrolysis-resistant phosphorothioate-modified analogs or PDE inhibitors. Suitable buffer systems for your special application have to be tested, but phosphate should be not optimal since one essential affinity interaction of cyclic nucleotides towards their target receptors is the cyclic phosphate. Regeneration can be achieved by a combination of up to 100 mM cAMP and buffer salts or 8 M urea.