INTRODUCTION Initial heating of a protein sample at 95°C in the presence of excess SDS and a thiol reagent denatures the protein mixture and disrupts disulfide bonds. Under these conditions, all reduced polypeptides bind the same amount of SDS (1.4 g of SDS per gram of polypeptide) independent of amino acid composition and sequence. The resolving power of SDS-PAGE is greatly enhanced by the inclusion of a "stacking gel," which uses the principles of isotachophoresis to concentrate samples into very small zones, but does not separate them. In the separating gel, the negatively charged SDS-protein complexes are separated solely on molecular-weight differences.