SDS-PAGE under focusing conditions: an electrokinetic transport phenomenon based on charge compensation.
A novel method is reported for mass separation of proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 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, called "SDS-PAGE focusing", exploits a "steady-state" process by which the SDS-protein micelles are driven to stationary zones along the migration path against a gradient of positive charges affixed to the neutral polyacrylamide matrix. As the total negative surface charge of such complexes matches the surrounding charge density of the matrix, the SDS-protein complex stops migrating and remains stationary, as typical of steady-state separation techniques. As a result of this mechanism, the proteins are separated in an unorthodox way, with the smaller proteins/peptides staying closer to the application point and larger proteins migrating further down toward the anodic gel end. This results in a positive slope of the Mr vs migration plot, vs a negative slope in conventional SDS-PAGE. Moreover, such a plot is linear (by design), whereas in standard SDS-PAGE it is semi- or even double logarithmic. Particularly advantageous appears the ability of the present method to fine-tune the separation of small-size fragments and tryptic digests, where conventional SDS-PAGE usually fails. Additionally, by exploiting constant plateaus of charges, rather than gradients, it is possible to amplify the separation between species having closely spaced Mr values, down to a limit of approximately 150 Da. This increases the resolution by at least 1 order of magnitude as compared with standard SDS-PAGE, where for a proper separation of two adjacent species, an Mr increment of approximately 3000 Da is needed.
Zilberstein
Gleb
G
Cleardirection Ltd., 4 Pekeris Street, Rehovot 76702, Israel.
Korol
Leonid
L
Antonioli
Paolo
P
Righetti
Pier Giorgio
PG
Bukshpan
Shmuel
S
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008823:Micelles; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D055672:Static Electricity
D008823:Micelles; D010446:Peptide Fragments; D011506:Proteins