Evaluation of capillary isoelectric focusing in glycerol‐water media with a view to hydrophobic protein applications

Abstract

Capillary isoelectric focusing (CIEF) separations are usually performed with neutral coated fused-silica capillaries in aqueous anticonvective media. Glycerol, a very viscous solvent (eta = 945 mPa x s at 25 degrees C), known to help stabilize any kind of proteins and solubilize hydrophobic ones, was tested as an alternative to using commercial gels. Viscosity and electroosmotic mobility were measured as a function of gel or glycerol content in water, and a 30:70 v/v glycerol-water medium appeared as a good compromise for performing CIEF in a bare fused-silica capillary without imposing too high a viscosity. To demonstrate the feasibility of this new CIEF system, a standard mixture of nine model proteins was separated according to their pI with a good agreement between experimental and literature aqueous pIs. Moreover, better resolution was achieved with this system than with the conventional aqueous CIEF system, as two of the model proteins could not be separated in the latter system. Glycerol-water CIEF in bare silica capillary was next applied to the separation of horse radish peroxidase, a complex mixture of protein isoforms. The good concordance with the separation obtained by the conventional CIEF system indicated the adequacy of this new system. Finally, as anticipated from the results obtained for the separation of bacteriorhodopsin, a membrane protein, glycerol-water CIEF performed in bare silica capillary appears to be a promising alternative to conventional aqueous CIEF for hydrophobic protein characterization, under their native form.

Use of quasi‐isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

“…As expected when using CIEF setup 3 no differences were observed between the different anolyte and catholyte couples. Moreover, with the HPC coating, the formamide plug remained very close to the anodic side: it covered less than 3 cm within 10 min, which corresponds to an electroosmotic velocity inferior to 10 25 cm 2 ?V 21 ?s 21 . In contrast, when using CIEF setup 1 the formamide plug moved towards the cathode as the focusing time increased, due to the presence of residual EOF.…”

“…In traditional fusedsilica capillaries, this is generally done by permanent or dynamic capillary coating. In the case of noncoated capillaries, the separation medium viscosity has then to be increased by glycerol [21] or methylcellulose [22] addition, which also limits EOF. Moreover, all these procedures avoid protein adsorption on capillary walls.…”

Capillary electrophoresis of proteins 2005–2007

“…The advantage of the system was demonstrated by analysis of a complex mixture of horseradish peroxidase isoforms and by analyzing hydrophobic membrane protein bacteriorhodopsin [43].…”

“…Viscosity and EOF were measured as a function of glycerol concentration and 30% glycerol was chosen as a good compromise. The system allowed better resolution of model proteins than CIEF in aqueous solutions [43].…”

Online CIEF‐ESI‐MS in glycerol–water media with a view to hydrophobic protein applications

“…(i) unattended preservation of electric continuity, (ii) the use of a MS compatible anticonvective medium can also help hydrophobic protein stabilization and (iii) the use of a bare fused-silica separation capillary that precludes the use of expensive and short-lifetime coated capillaries. Indeed, in a previous work [45], we demonstrated the feasibility of performing CIEF in glycerol-water media and in uncoated capillaries. Thanks to its high viscosity, glycerol, which is known to better solubilize and stabilize hydrophobic proteins, can both play the role of anticonvective medium and allow employing bare silica capillaries, as it considerably reduces EOF.…”