Protein adsorption in fused-silica and polyacrylamide-coated capillaries.
The model proteins cytochrome c, myoglobin, ovalbumin, and beta-lactoglobulin were investigated with regard to their adsorption properties on capillaries for electrophoresis. The model compounds were selected to cover a wide range of properties. Cytochrome c is a basic protein (isoelectric point (pI): 9.6; M(r): 11.7 kDa), beta-lactoglobulin is rather acidic (pI: 5.4, M(r): 18.4 kDa), myoglobin was chosen as a neutral reference protein (pI: 6.8-7.4, M(r): 17.8 kDa), and ovalbumin (pI: 5.1, M(r): 45.0 kDa) was selected as a relatively larger analyte. First, the pH dependence of adsorption was investigated for the bare fused silica. A clear correlation to the respective pIs was noted. For myoglobin and ovalbumin, none or negligible adsorption was found above the pI, whereas strong adsorption was noted just below this parameter. Cytochrome c and beta-lactoglobulin already showed distinct adsorption above their pIs. However, none of the proteins showed any significant adsorption more than one pH unit above the pIs. For linear polyacrylamide-coated capillaries, a decreased but not a complete lack of adsorption was observed. Here, pH-dependent adsorption was noted as well. Regeneration of the capillaries by rinsing with buffers containing 200 mM SDS was also investigated. This method was completely successful for myoglobin, but that too for only freshly-adsorbed protein. After a storage time of 24 h and due to the aging of the adsorbate, a sufficient regeneration was no longer possible.
Graf
Michael
M
Institute of Pharmacy, TU Braunschweig, Braunschweig, Germany.
Galera García
Raquel
R
Wätzig
Hermann
H
D000327:Adsorption; D045304:Cytochromes c / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007782:Lactoglobulins / Q000302:isolation & purification*; D009211:Myoglobin / Q000302:isolation & purification*; D010047:Ovalbumin / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate
D007782:Lactoglobulins; D009211:Myoglobin; D012967:Sodium Dodecyl Sulfate; D010047:Ovalbumin; D045304:Cytochromes c