Transverse urea-gradient gel electrophoresis

Current Protocols in Protein Science
D P Goldenberg

Abstract

Monitoring the cooperative unfolding transition induced when a protein is exposed to elevated temperature or a chemical denaturant is an important strategy for characterizing the conformational properties of a globular protein. This transition may be analyzed quantitatively by a variety of spectroscopic techniques, but a simpler alternative is described in this unit: urea-gradient gel electrophoresis. The pattern produced in the resulting gel can be used to estimate both the free energy change for unfolding and the rate of the unfolding transition. In addition, the technique can help identify either covalent or conformational heterogeneity in a protein sample. Because urea-gradient gel patterns are sensitive to several parameters, including hydrodynamic volume, net charge, and conformational stability, the technique can be particularly useful for comparing two forms of a protein, e.g., a natural form and the product of recombinant bacteria.

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