Practice question

Electrophoretic Mobility Shift Assay is a classical in vitro method for analyzing DNA-protein interaction. A radiolabeled or fluorescent DNA probe containing putative binding site is incubated with purified protein or nuclear extract, then resolved on non-denaturing polyacrylamide gel. Free DNA migrates rapidly due to high negative charge. DNA bound to protein gains increased mass, reduced charge-to-mass ratio and conformational change, causing retarded mobility. Appearance of shifted band demonstrates specific physical association. Competitor DNA tests specificity, while titration estimates affinity. It is unsuitable for evaluating protein folding or solubility.