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.
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.
Radko
Sergey P
SP
Section on Macromolecular Analysis, National Taiwan University, Taipei.
Chang
Huan-Tsung
HT
Zakharov
Sergey F
SF
Bezrukov
Ludmila
L
Yergey
Alfred L
AL
Vieira
Nancy E
NE
Chrambach
Andreas
A
Chen
Huan-Tsung
HT
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007525:Isoelectric Focusing; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014462:Ultrafiltration
D011506:Proteins