An attempt to resolve all the various proteins in a single human cell type by two-dimensional electrophoresis: I. Extraction of all cell proteins.
A concept is presented for estimation of the total number of different proteins in a single human cell type (exemplified here by Hep cells) by use of two-dimensional electrophoresis (2DE). This concept includes three problems, the first, investigated in this study, being the transfer of all protein species of the cells into a sample useful for separation by 2DE. Five different extraction media containing--in various combinations--urea, Nonidet P-40, Zwittergent, mercaptoethanol, dithiothreitol, and sodium dodecyl sulfate were used step by step in three different extraction procedures to extract the cell proteins. The amount of radiolabeled proteins in each extract was measured. Each extract was subjected to 2DE. From the total mass of cell proteins, 99.99% could be extracted in two steps: 96% were extracted with urea/beta-mercaptoethanol solution, the remaining 4% with sodium dodecyl sulfate/urea/beta-mercaptoethanol solution. A special class of proteins assumed to be present in the latter fraction was not detected. Thus this fraction can be omitted from the further analysis of all cell proteins by 2DE. Protein classes that possibly remain undetected by the described extraction procedures are mentioned.
D002460:Cell Line; D004229:Dithiothreitol; D004586:Electrophoresis; D006801:Humans; D007525:Isoelectric Focusing; D007822:Laryngeal Neoplasms / Q000032:analysis; D008623:Mercaptoethanol; D017830:Octoxynol; D011092:Polyethylene Glycols; D011506:Proteins / Q000302:isolation & purification*; D000644:Quaternary Ammonium Compounds; D012967:Sodium Dodecyl Sulfate; D014508:Urea
D011506:Proteins; D000644:Quaternary Ammonium Compounds; C034730:zwittergent 3-12; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D008623:Mercaptoethanol; D014508:Urea; D017830:Octoxynol; C010615:Nonidet P-40; D004229:Dithiothreitol