OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins

摘要: In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on use sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study reagents used for PAGE, OFFGEL-IEF separation, post-separation treatment gels (such as enzymatic digestion sample preparation MS analysis) was tackled regarding their suitability identification platinum binding proteins using standard incubated with cisplatin. distribution in high low molecular weight fractions (separated by cut-off filters) determined ICP-MS, which allows evaluating bond stability under conditions studied. SDS-PAGE absence β-mercaptoethanol or dithiotreitol preserved bonds. addition, neither influence electric field during nor processes fixing, staining destaining gel did result loss from proteins. non-reducing provides platinum-binding very narrow bands quantitative recoveries. Different amounts platinum-bound covering range 0.3-2.0 μg were separated mineralised determination, showing good linearity. Limits detection mixture five cisplatin between 2.4 13.9 pg platinum, satisfactory application biological samples. Regarding OFFGEL-IEF, denaturing solution without thiourea recommended. kidney cytosol demonstrated.