Practice question

In EMSA, labeled DNA probe is subjected to non-denaturing gel electrophoresis maintaining native complexes. Free unbound DNA, being small and highly negatively charged, migrates rapidly toward positive electrode, appearing near bottom of autoradiogram. When sequence-specific DNA-binding protein present, DNA engages in stable nucleoprotein complex with substantially larger hydrodynamic radius and altered charge-to-mass ratio. Consequently, electrophoretic mobility is retarded, complex migrates slower and appears as higher band. Intensity and position of retarded band reflect affinity, stoichiometry, and specificity of interaction, which can be confirmed by competition with unlabeled cold probe.