title: Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis.
abstract: We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.
authors.affiliation: Australian Proteome Analysis Facility, School of Biological Sciences, Macquarie University, Sydney, NSW, Australia.
authors.forename: M P
authors.initials: MP
authors.lastname: Molloy
authors.forename: B R
authors.initials: BR
authors.lastname: Herbert
authors.forename: B J
authors.initials: BJ
authors.lastname: Walsh
authors.forename: M I
authors.initials: MI
authors.lastname: Tyler
authors.forename: M
authors.initials: M
authors.lastname: Traini
authors.forename: J C
authors.initials: JC
authors.lastname: Sanchez
authors.forename: D F
authors.initials: DF
authors.lastname: Hochstrasser
authors.forename: K L
authors.initials: KL
authors.lastname: Williams
authors.forename: A A
authors.initials: AA
authors.lastname: Gooley
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D008565:Membrane Proteins / Q000302:isolation & purification*; D012995:Solubility; D012996:Solutions
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D012996:Solutions