Asunto(s)
Citocinas/química , Portadores de Fármacos/química , Glutatión Transferasa/metabolismo , Ácido Hialurónico/química , Hialuronoglucosaminidasa/metabolismo , Hidrogeles/química , Polietilenglicoles/química , Animales , Citocinas/administración & dosificación , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/metabolismo , Portadores de Fármacos/administración & dosificación , Liberación de Fármacos , Humanos , Interleucina-4/química , Ratones , Modelos Animales , Compuestos de Sulfhidrilo/químicaRESUMEN
A novel assay was developed for the simultaneous monitoring of quantum dots and their assembly and disassembly with PreScission protease using capillary electrophoresis with fluorescence detection. Quantum dots and PreScission protease were injected into a capillary sequentially, then mixed and assembled via a thioether bond upon coupling to glutathione S-transferase tag inside the capillary. The in-capillary assembly was influenced by the molar ratio and the time interval of injection. Furthermore, the simultaneous monitoring of quantum dots and their assembly with PreScission protease and glutathione induced disassembly was achieved by adjusting the sampling sequence and the time interval of injection. More importantly, the in-capillary assay could be also applied to the online detection of glutathione.
Asunto(s)
Glutatión Transferasa/química , Péptido Hidrolasas/química , Puntos Cuánticos , Electroforesis Capilar , Glutatión Transferasa/metabolismo , Microscopía Fluorescente , Péptido Hidrolasas/metabolismoRESUMEN
Here, we present protocols describing the use of the dipeptidyl-aminopeptidase-1 (DPP1, DAPase) exoprotease-based TAGZyme system and the endoprotease, Factor Xa. Both enable the recovery of proteins free of any amino acids encoded by the vector and/or protease recognition site. They also provide the possibility of removing the proteases from the preparation of the target protein by a simple subtractive chromatography step. TAGZyme enzymes contain an uncleavable His tag for removal by Immobilized Metal Ion Affinity Chromatography (IMAC). Factor Xa can be removed using Xa Removal Resin.