Third generation of focusing: gel matrices with immobilized cation gradients.

A novel method is reviewed here for separation of polyanions, based not on conventional zone electrophoretic means, but on a "steady-state" process by which said polyanions 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 polyanions matches the surrounding charge density of the matrix, they stop migrating and remain stationary, as typical of steady-state separation techniques. This technique has been successfully applied to SDS-protein micelles, DNAs, RNAs and heparins, with remarkable separations, often much superior than those obtained in conventional techniques.

Steady-state electrophoresis of RNA against a gradient of cationic charges in a polyacrylamide matrix.

A novel method for separation of RNA fragments is reported here, based on migrating the polyanionic RNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations are typically performed in a 0-10 mM, pK 10.3 Immobiline gradient under denaturing conditions (6 M urea).

High-resolution separation of peptides by sodium dodecyl sulfate-polyacrylamide gel "focusing".

As a follow-up of our previous report (Anal. Chem. 2007, 79, 821-827) on analytical SDS-PAGE focusing, a refinement of the method for separation of peptides in the small to medium M(r) range (0.

DNA separation methodology based on charge neutralization in a polycationic gel matrix.

A novel method for separation of DNA fragments is here reported, based on migrating the polyanionic DNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated under two working conditions: either against a gradient of positive charges, to allow the various DNA fragments to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization, or in a plateau gel (i.e.

SDS-PAGE focusing: preparative aspects.

As a followup of our previous report (Zilberstein, G.; Korol, L.; Antonioli, P.

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.

Parallel isoelectric focusing II.

A miniature electrophoretic device is developed on the basis of a new isoelectric focusing (IEF) method, namely parallel isoelectric focusing. We report here the theory and the results of operation of a new parallel isoelectric device (PID). The main advantages and limitations of the method are discussed for miniaturization purposes.

Parallel isoelectric focusing chip.

Fast isoelectric focusing (IEF) is becoming a key method in modern protein analysis. We report here the theory and experimental results of new parallel isoelectric devices (PID) for fast IEF. The main separation tool of any PID is a dielectric membrane with conducting channels filled by immobiline gels of varying pH.

Parallel processing in the isoelectric focusing chip.

Investigation of isoelectric focusing (IEF) kinetics has been performed to provide the theoretical basis for miniaturization of classical IEF in immobilized pH-gradients. Standard IEF demands colinearity of the electric field and pH-gradient directions (serial devices). It is shown that the IEF separation process based on a continuous, serial pH gradient is incompatible with miniaturization of separation devices.