Abstract
A polyacrylamide-gel-electrophoresis method has been developed that permits the analysis of conformational changes that occur during the thermal denaturation of macromolecules. A stable transverse temperature gradient was produced in an aluminium heating jacket clamped around a vertical polyacrylamide slab gel. After temperature equilibration, gels were loaded with either a layer of protein solution (20-200 micrograms/gel) or a solution of double-stranded DNA (20 micrograms/gel) and electrophoresis begun. At the end of the run the gels were stained and the effect of temperature on mobility observed. The technique proved informative both for the irreversible unfolding of proteins (Drosophila alcohol dehydrogenase and lactic acid dehydrogenase) and for a protein that was reversibly denatured by heat (beta-lactamase). In the latter case a clear transition between the native enzyme and a slower-migrating denatured state was observed. The patterns obtained were analogous to the type produced by the transverse-urea-gradient-electrophoretic method of Creighton [(1979) J. Mol. Biol. 129, 253-264]. The method also resolved a complex mixture of double-stranded-DNA restriction-digest fragments.