title: High-efficiency capillary isoelectric focusing of protein complexes from Escherichia coli cytosolic extracts.
abstract: High-efficiency capillary isoelectric focusing (cIEF) separations of protein complexes obtained from soluble protein fractions are demonstrated. Size-exclusion chromatography was used as a first dimension separation to fractionate putative protein complexes with apparent molecular masses of up to 1,500,000 from an Escherichia coli cytosolic fraction. Non-denaturing cIEF separations using highly hydrophilic polymer-coated capillaries constituted the second dimension. The conditions developed produced reproducible and high-efficiency separations, corresponding to approximately 2 x 10(6) theoretical plates and peak capacities of approximately 10(3) for pH 3-10 cIEF separations in 65 cm long capillaries. Combination of the two non-denaturing separation dimensions permitted isolation and analysis of individual protein complexes from complicated biological samples. Studies indicated that many E. coli complexes were stable on the time scale of the cIEF separations, but were degraded upon more extended periods of storage on ice, necessitating rapid sample processing and fast analysis techniques.
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Shen
authors.forename: S J
authors.initials: SJ
authors.lastname: Berger
authors.forename: R D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D007525:Isoelectric Focusing / Q000379:methods*; D008970:Molecular Weight
chemical_list: D001426:Bacterial Proteins