EXTRACTION OF MEMBRANE-PROTEINS BY DIFFERENTIAL SOLUBILIZATION FOR SEPARATION USING 2-DIMENSIONAL GEL-ELECTROPHORESIS

We describe the extraction and enrichment of membrane proteins for sep aration by two-dimensional polyacrylamide gel electrophoresis (2-D PAG E) after differential solubilization of an Escherichia coli cell lysat e. In a simple three-step sequential solubilization protocol applicabl e for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally u sed for isoelectric focusing (IEF). As the first step, Tris-base was u sed to solubilize many cytosolic proteins. The resultant pellet was th en subjected to conventional solubilizing solutions (urea, 3-[(3-chola midopropyl) dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of th e initial E. coli sample mass was solubilized. Finally, the membrane p rotein rich pellet was partially solubilized using a combination of ur ea, 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 o uter membrane proteins (Omp), OmpW and OmpX, have previously been know n only as an open reading frame in E. coli, while OmpC, OmpT and OmpTO LC have not previously been identified on a 2-D gel. The prefractionat ion of an entire cell lysate into multiple fractions, based on solubil ity, results in simplified protein patterns following 2-D PAGE using b road-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advan tages of sample prefractionation are that protein identification and g el matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extra ction is conducted in a single centrifuge tube, minimizing protein los s.