Practice question

In EMSA, electrophoretic mobility under non-denaturing conditions reflects size and charge. Free double-stranded DNA probe migrates quickly toward anode owing to phosphate backbone negativity. When sequence-specific DNA-binding protein recognizes probe, resulting complex possesses substantially higher molecular weight, altered conformation and lower charge density, causing markedly slower migration. Consequently, complex appears as higher, slower band compared to free probe. This retarded band confirms formation of DNA-protein complex, useful for transcription factor analysis. DNA degradation would create fast smear, contamination alone would not yield discrete specific shift.