A quantitative cryogenic gel-shift technique for analysis of protein-DNA binding.

A cryogenic gel mobility shift technique was developed in which a mixture of protein and DNA samples at equilibrium is rapidly quenched and electrophoresed at -40 degrees C. The rapid and sustained drop in temperature results in almost complete stabilization of the equilibrium species distribution. Autoradiogram analysis of relative abundances for the bound and free DNA sites is carried out over a range of initial binding ratios to yield the binding curve and equilibrium constant as in the usual gel-shift assay. Validity of this technique for determining equilibrium populations of the interacting species is based upon two testable assumptions: (i) The equilibrium species distribution does not change during the cryogenic quench procedure. (ii) This equilibrium distribution is also constant during electrophoresis of the sample. Evidence supporting these assumptions was obtained using lambda cI repressor and a 570-bp DNA fragment containing the repressor binding site OR1. The resolved free energy for this interaction (delta G1) was shown to be independent of the quench procedure, duration of the quench stage, residence time in the gel wells, and duration of low-temperature electrophoresis. The technique yielded a free energy that was in close agreement with those from filter binding and DNAse footprint titration methods. This cryogenic version of the gel-shift method may prove especially useful in cases like that of lambda cI/OR1 binding, for which conventional gel-shift methodology has not been feasible.