| Ornithine decarboxylase: Expression and regulation in rat brain and in transgenic mice | ||
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Sprague-Dawley male rats were killed by decapitation. The brains were carefully removed and frozen at –70 ¢ªC for one hour. Dissections were performed on an ice-cooled glass plate by the method of Glawinski and Iversen (Glowinski & Iversen 1966). The brain region were stored at –70 ¢ªC until used and then homogenised in 3 vol. of cold 25 mM Tris-HCl buffer, pH 7.4, containing 0.1 mM EDTA, 5 mM dithiothreitol (DTT) and 0.1 mM pyridoxal 5’-phosphate (PLP). The homogenates were centrifuged at 105 000 x g for one hour at 4 ¢ªC. After double (NH4)2SO4 precipitation (20 % - 60 %) the pellets were dissolved in 0.1 M Tris-HCl buffer, pH 7.1, containing 4 mM EDTA, 4 mM DTT and 0.4 mM PLP.
The dissolved pellets were treated with 250 mM NaCl and applied to a Sephadex G-75 Superfine column (Pharmacia Biotech AB, Uppsala, Sweden) to separate ODC and antizyme. The column was eluated with 0.1 M Tris-HCl buffer, pH 7.1, containing 4 mM EDTA, 4 mM DTT, 0.4 mM PLP and 250 NaCl. After gelfiltration NaCl, which decreases ODC activity in assay mixture (Kallio et al. 1979), was removed from fractions using Sephadex G-25 columns (PD-10 columns, Pharmacia Biotech AB, Uppsala, Sweden). Desalted fractions were used for ODC and antizyme activity assays.