Gel protein capillary extraction apparatus. electronic protein transfer.
A gel protein capillary extraction apparatus is developed and demonstrated for its rapid and effective transfer of SDS-protein complexes from polyacrylamide gel to a fused-silica capillary. The small dimensions of capillary columns permit the application of high voltages for achieving rapid and effective transfer of gel proteins. Furthermore, the fused-silica capillaries are internally coated with polyacrylamide for the elimination of electroosmotic pumping and protein adsorption onto the capillary wall. The extracted proteins are present in a highly concentrated solution plug as the result of field amplification and sample stacking during the extraction process. Three model proteins, including cytochrome c (14 kDa), ovalbumin (45 kDa), and beta-galactosidase (116 kDa), are visualized using coomassie blue staining and electrophoretically extracted from the gels with protein loading as low as 50 ng. The SDS-cytochrome c complexes extracted from a 50-ng protein loading are concentrated in a 30-nL solution plug inside the capillary with an estimated concentration of 0. 1 mg/mL or 10(-5) M. The capillary format allows the straightforward integration of a miniaturized trypsin-membrane reactor for on-line proteolytic digestion and ESI-MS analysis for protein/peptide identification.
Cooper
Jonathan W
JW
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
Gao
Jun
J
Lee
Cheng S
CS
D002138:Calibration; D003574:Cytochrome c Group / Q000737:chemistry; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D007202:Indicators and Reagents; D011506:Proteins / Q000302:isolation & purification*
D003574:Cytochrome c Group; D005782:Gels; D007202:Indicators and Reagents; D011506:Proteins