Premium
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
Author(s) -
Radko Sergey P.,
Chen HuanTsung,
Zakharov Sergey F.,
Bezrukov Ludmila,
Yergey Alfred L.,
Vieira Nancy E.,
Chrambach Andreas
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200204)23:7/8<985::aid-elps985>3.0.co;2-r
Subject(s) - electroelution , isoelectric focusing , chromatography , chemistry , sodium dodecyl sulfate , molecular weight size marker , gel electrophoresis , polyacrylamide gel electrophoresis , two dimensional gel electrophoresis , electrophoresis , isoelectric point , ultrafiltration (renal) , immobilized ph gradient , gel electrophoresis of proteins , mass spectrometry , biochemistry , proteomics , gene , enzyme
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 νg upon illumination by a green laser and 0.5–1 νg 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.