Mp. Molloy et al., EXTRACTION OF MEMBRANE-PROTEINS BY DIFFERENTIAL SOLUBILIZATION FOR SEPARATION USING 2-DIMENSIONAL GEL-ELECTROPHORESIS, Electrophoresis, 19(5), 1998, pp. 837-844
Citations number
18
Categorie Soggetti
Biochemical Research Methods","Chemistry Analytical
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.