Separation of plasma membrane proteins of cultured human fibroblasts by affinity chromatography on bonded microparticulate silicas.
Adsorbents for high-performance affinity chromatography were prepared by bonding proteins and reactive Procion triazine dyes to 3-isothiocyanatopropyl- and 3-aminopropylsilicas. The materials prepared were used successfully in the separation of hydrophobic plasma membrane proteins of cultured human fibroblasts. The data obtained show that the reaction of 3-isothiocyanatopropyltriethoxysilane (ITCPS) with the surface hydroxyl groups of silica yields a new and convenient route to preparing an "activated carrier" that is capable of coupling with potential affinity ligands containing amino functional groups. The reaction and bonding procedures of 3-isothiocyanatopropyltriethoxysilane and 3-aminopropyltriethoxysilane with silica were optimized with regard to a controlled and reproducible ligand density by adjusting the type of solvent and organic base as reaction catalyst. The ligand content of reactive triazine dyes directly coupled to 3-aminopropylsilica was significantly higher than that of the 6-aminohexyl derivatives coupled to 3-isothiocyanatopropylsilica. The stability of Procion Blue MX-R bonded to 3-aminopropylsilica and 3-isothiocyanatopropylsilica in phosphate-buffered aqueous solution at pH 5.0 and 8.0 was examined.
D002462:Cell Membrane / Q000032:analysis*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents / Q000032:analysis; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017879:Isothiocyanates; D008024:Ligands; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D012821:Silanes; D058428:Silica Gel; D012822:Silicon Dioxide / Q000032:analysis
D004396:Coloring Agents; D003902:Detergents; D017879:Isothiocyanates; D008024:Ligands; D008565:Membrane Proteins; D012821:Silanes; C042856:3-isothiocyanatopropyltriethoxysilane; D058428:Silica Gel; D012822:Silicon Dioxide