Two-dimensional polyacylamide gel electrophoresis of envelope proteins of Escherichia coli.
A method of separating envelope proteins by two-dimensional polyacrylamide gel electrophoresis is described. Escherichia coli envelopes (inner and outer membranes) were prepared by French pressing and washed by repeated centrifugation. Membrane proteins were solubilized with guanidine thiocyanate and were dialyzed against urea prior to two-dimensional electrophoretic analysis. The slab gel apparatus and conditions were similar to the technique developed by Metz and Bogorad (1974) for the separation of ribosomal proteins. This separation occurs in 8 M urea for the first dimension and in 0.2% sodium dodecyl sulfate for the second dimension. The technique separates about 70 different membrane proteins in a highly reproducible fashion according to both intrinsic charge and molecular weight. Some examples of alterations in the membrane protein pattern are demonstrated. These alterations are caused by a mutation affecting a sugar transport system and by growth in the presence of D-fucose, inducer of the transport system. A further example of membrane protein changes introduced by growth at the nonpermissive temperature of a temperature-sensitive cell division mutant is shown. Finally, it is demonstrated that the major outer membrane component of Escherichia coli K-12 contains more than four proteins of similar molecular weight.
D001426:Bacterial Proteins / Q000032:analysis*; D002455:Cell Division; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis; D002473:Cell Wall / Q000032:analysis; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000032:analysis* / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D005643:Fucose / Q000378:metabolism; D005696:Galactosidases / Q000378:metabolism; D006146:Guanidines; D005779:Immunodiffusion; D008756:Methylgalactosides / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D012967:Sodium Dodecyl Sulfate; D012997:Solvents; D013696:Temperature; D013861:Thiocyanates; D014508:Urea
D001426:Bacterial Proteins; D006146:Guanidines; D008756:Methylgalactosides; D012997:Solvents; D013861:Thiocyanates; D005643:Fucose; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D005696:Galactosidases