Electroelution without gel sectioning of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Fluorescent detection, recovery, isoelectric focusing and matrix assisted laser desorption/ionization-time of flight of the electroeluate

Abstract

A method of direct electroelution of intact proteins, without gel sectioning and orthogonal to the orientation of electrophoretic migration, was developed in application to Novex gels, using a simple home-made experimental setup. Six model proteins covering the molecular mass range of 14-120 kDa were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with an aqueous solution of the fluorescent dye, SYPRO-red, and electroeluted from the intact gel. The sensitivity of visual detection was 0.1-0.2 microg upon illumination by a green laser and 0.5-1 microg of protein on side-ways UV-illumination. Duration (for each protein) and field strength were optimized to render protein electroelution from the gel near-quantitative (above 80%) and relatively fast (1-12 min at 1 kV). At a given field strength, the optimal duration was found to be inversely proportional to the mobility of proteins in SDS-PAGE. Sequential ultrafiltration was evaluated as a simple approach to concentrate electroeluted proteins and deplete SDS to a level compatible with mass spectrometry of proteins: protein yields in the electroeluate were 25-33% (depending on the protein used) after three steps of ultrafiltration with water. The analysis of the electroeluate by isoelectric focusing in an immobilized pH gradient, to reveal protein heterogeneity under a single SDS-PAGE band (prior, e.g., to mass spectrometric analysis), was demonstrated.

Rapid protein separations in ultra-short microchannels: microchip sodium dodecyl sulfate–polyacrylamide gel electrophoresis and isoelectric focusing

“…To visualize all three peaks at once, multiple fluorescence images taken at different excitation wavelengths were merged into a single image electronically. The two peaks observed for the carbonic anhydrase II protein are charge isomers of the same protein, as observed previously [25].…”

SDS-PAGE Focusing: Preparative Aspects

“…SDS-PAGE, perhaps the most popular electrophoretic technique for rapid assessment of protein purity and M r values, also appears as a promising technique for microscale preparative runs. Disregarding past attempts at eluting protein zones via cutting out of stained zones, recent methodologies have focused on the possibility of direct electroelution of proteins from SDS-PAGE gels without gel sectioning. − While such techniques could be applied to both 1D and 2D gels, a shotgun electroelution system, driving a multitude of proteins spots resolved in a 2D surface into a multichambered, gridlike layer composed of as many as 384 wells has been described, as a proteomic tool for large-scale analysis of resolved polypeptide chains at the end of a 2D mapping process …”

A novel microscale preparative gel electrophoresis system

“…20,21 Buzás 20 compared the protein recovery using this apparatus and in-gel digestion, and the results indicated that this apparatus produced a high recovery. Radko 21 studied various aspects of this application including visualized detection, eluted protein recovery, IEF of the electroeluate and MALDI-TOF-MS of SDS-proteins after elution and ultrafiltration. This electroelution apparatus is capable of fast elution but only permits the elution of a single band at a time.…”