Selective extraction of outer-membrane proteins from membrane complexes of Pseudomonas maltophila by chloroform-methanol.
An organic phase partitioning method is described for the selective purification of outer-membrane proteins (OMPs) from the total membrane complex of the opportunistic human and sheep pathogen, Pseudomonas maltophila. SDS-PAGE analysis confirmed that OMPs purified by chloroform-methanol treatment of the total membrane complex were not only identical to OMPs extracted from outer-membrane vesicles separated by sucrose gradient density centrifugation, but also possessed little or non-detectable levels of inner-membrane contaminants. Further analysis by enzyme linked immunosorbent assay (ELISA) and immunoblotting established that OMPs extracted by organic phase partitioning with chloroform-methanol retained antigenicity and serological activity indistinguishable from OMPs that were present in outer-membrane vesicles resolved by isopycnic sucrose density centrifugation of sarkosyl-treated membrane complexes.
Chin
J
J
Central Veterinary Laboratory, New South Wales Department of Agriculture, Glenfield, Sydney, Australia.
Dai
Y
Y
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D002725:Chloroform; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D000432:Methanol; D011549:Pseudomonas / Q000032:analysis*; D012756:Sheep
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D002725:Chloroform; D000432:Methanol