title: Activity staining of glutathione peroxidase after two-dimensional gel electrophoresis.
abstract: We have developed a method for rapid activity staining of proteins with glutathione peroxidase (GPx) activity after 2-D gel electrophoresis. After separating proteins extracted from yeast, or mouse red blood cells, by two-dimensional gel electrophoresis, SDS was removed and the gel was submerged in a Tris-HCl buffer containing glutathione and hydrogen peroxide, followed by incubation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and phenazine methosulfate (PMS). After this proteins with GPx activity appeared as clear zones on a purple background. This relatively simple activity staining method could be useful for rapid screening of proteins with GPx activity in cell extracts.
authors.affiliation: Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-600, Korea.
authors.forename: Chang Won
authors.initials: CW
authors.lastname: Kho
authors.forename: Sung Goo
authors.initials: SG
authors.lastname: Park
authors.forename: Do Hee
authors.initials: DH
authors.lastname: Lee
authors.forename: Sayeon
authors.initials: S
authors.lastname: Cho
authors.forename: Goo Taeg
authors.initials: GT
authors.lastname: Oh
authors.forename: Seongman
authors.initials: S
authors.lastname: Kang
authors.forename: Byoung Chul
authors.initials: BC
authors.lastname: Park
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D004396:Coloring Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D005979:Glutathione Peroxidase / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D008623:Mercaptoethanol / Q000494:pharmacology; D008773:Methylphenazonium Methosulfate / Q000494:pharmacology; D051379:Mice; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D004396:Coloring Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D008773:Methylphenazonium Methosulfate; D008623:Mercaptoethanol; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase; C022616:thiazolyl blue; D005978:Glutathione; D004229:Dithiothreitol