Protocol

SDS-PAGE of Proteins

This protocol was adapted from "Concentrating Solutions of Protein," in Appendix 5 of Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

The first 100 words of the full text of this article appear below.

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 . . . [Full Text of this Article]

MATERIALS

Reagents

Equipment

METHOD