EC Number   |
|---|
   1.14.11.13 | - |
| 1.14.11.13 | 24 h after application of 14C-labelled substrate to the shoot apex, whole shoots are harvested, rinsed with water, same genotype shoots combined and homogenized in 80% methanol water, stirring overnight at 4°C, filtration, residue re-extracted in methanol for 1 h, filtered, combined filtrates vacuum dried, weak acid fraction prepared by solvent partitioning, anion exchange, and C18 SPE (no solvent partitioning for substrate gibberellin 9), purified samples are analyzed by reverse-phase HPLC with online radiomonitoring, identification of radiolabeled products by GC-MS |
| 1.14.11.13 | Escherichia coli cells with recombinant enzyme grown at 37°C, harvested and suspended in lysis buffer (100 mM Tris-HCl, pH 7.5), sonication in ice water, centrifuged, supernatant collected and stored at -80°C for enzyme activity assays |
| 1.14.11.13 | Escherichia coli with recombinant enzyme are centrifuged, resuspended in a BugBuster Protein Extraction Reagent, centrifuged, supernatants eluted through a GST-Bind resin with 50 mM Tris buffer, pH 8.0, containing 10 mM reduced glutathione, storage at -80°C |
| 1.14.11.13 | glutathione agarose bead affinity chromatography and gel filtration |
| 1.14.11.13 | glutathione Sepharose 4B column chromatography |
| 1.14.11.13 | recombinant enzyme |
| 1.14.11.13 | Sephadex G-25 column chromatography and Superdex G-25 column chromatography, Mini S column chromatography and gel filtration chromatography |
| 1.14.11.13 | two gibberellin 2beta-hydroxylases partially purified |
