Influence of sample preparation technique on two-dimensional gel electrophoresis of proteins from Porphyromonas gingivalis.
Sample preparation methods were compared for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of cellular proteins from the proteolytic bacterium Porphyromonas gingivalis. Standard solubilization buffer yielded poorly resolved protein spots, but pre-treatment of cells with trichloroacetic acid or inclusion of the protease inhibitor TLCK during solubilization improved definition and separation. The latter approach allowed reliable detection of a 55 kDa immunodominant surface antigen by Western immunoblotting. Further improvements in resolution occurred when SDS was included in the sample preparation. Thus, controlling proteolysis and optimizing protein solubilization were essential for reproducible separations and maximal protein recovery during 2D-PAGE of P. gingivalis.
Pridmore
A M
AM
Division of Oral Biology, Leeds Dental Institute, University of Leeds, Shipley, UK.
Devine
D A
DA
Bonass
W A
WA
Silley
P
P
D001426:Bacterial Proteins / Q000032:analysis*; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D016966:Porphyromonas gingivalis / Q000737:chemistry* / Q000254:growth & development
D001426:Bacterial Proteins