A mobility shift assay is electrophoretic separation of a protein–DNA or protein–RNA mixture on a polyacrylamide or agarose gel for a short period (about 1.5-2 hr for a 15- to 20-cm gel). The speed at which different molecules (and combinations thereof) move through the gel is determined by their size and charge, and to a lesser extent, their shape (see gel electrophoresis). The control lane (DNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. However, assuming that the protein is capable of binding to the fragment, the lane with a protein that binds present will contain another band that represents the larger, less mobile complex of nucleic acid probe bound to protein which is shifted up on the gel (since it has moved more slowly).
Under the correct experimental conditions, the interaction between the DNA (or RNA) and protein is stabilized and the ratio of bound to unbound nucleic acid on the gel reflects the fraction of free and bound probe molecules as the binding reaction enters the gel. This stability is in part due to a "caging effect", in that the protein, surrounded by the gel matrix, is unable to diffuse away from the probe before they recombine. If the starting concentrations of protein and probe are known, and if the stoichiometry of the complex is known, the apparent affinity of the protein for the nucleic acid sequence may be determined. Unless the complex is very long lived under gel conditions, or dissociation during electrophoresis is taken into account, the number derived is an apparent Kd.  If the protein concentration is not known but the complex stoichiometry is, the protein concentration can be determined by increasing the concentration of DNA probe until further increments do not increase the fraction of protein bound. By comparison with a set of standard dilutions of free probe run on the same gel, the number of moles of protein can be calculated.