Activity staining of glutathione peroxidase after two-dimensional gel electrophoresis.
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.
Kho
Chang Won
CW
Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-600, Korea.
Park
Sung Goo
SG
Lee
Do Hee
DH
Cho
Sayeon
S
Oh
Goo Taeg
GT
Kang
Seongman
S
Park
Byoung Chul
BC
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
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