SDS-PAGE focusing: preparative aspects.
As a followup of our previous report (Zilberstein, G.; Korol, L.; Antonioli, P.; Righetti, P. G.; Bukshpan, S. Anal. Chem. 2007, 79, 821-827) on analytical SDS-PAGE focusing, a novel method is here reported for small-scale prefractionation of complex protein mixtures, for subsequent proteome analysis, based on mass separation of SDS-protein micelles not in a gel matrix, but in liquid cationic polymers assembled in a multicompartment electrolyzer (MCE) in a stepwise fashion at discrete and increasing levels of positive charges (from 3 to 28 mM), the neighboring chambers being separated by neutral agarose membranes. Unlike conventional SDS-PAGE, in which separation by mass of SDS-laden polypeptide chains is obtained in constant concentration or porosity gradient gels, the present method of SDS-PAGE focusing exploits a "steady-state" process by which the SDS-protein micelles are driven to stationary zones along the migration path and trapped into different compartments of the MCE device via interaction (and subsequent charge neutralization) with cationic polymers of fixed (but increasing from chamber to chamber from cathode to anode) charge density. Minimization of migration of the liquid cationic polymers is obtained via use of low voltage and by arranging for a buffer conductivity gradient along the migration path. The present setup has the advantage of high protein recoveries (up to 90%) without any contamination from ungrafted monomers and catalysts, as occurring in proteins recovered by passive elution from gel matrixes. Additionally, resolution can be fine-tuned by selecting cationic polymers of varying charge density in microstep increments. The cationic polymers, of desired charge density and proper viscosity, are prepared by standard polymerization conditions, can be easily precipitated and washed free of monomeric contaminants, and stored in a dry form for subsequent use.
Zilberstein
Gleb
G
Cleardirection Ltd., 4 Pekeris Street, Rehovot 76702, Israel.
Korol
Leonid
L
Righetti
Pier Giorgio
PG
Bukshpan
Shmuel
S
D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D011506:Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011506:Proteins