Electrophoretic mobility shift assay (EMSA)

Introduction

• Advantage
  • can separate different types of complexes, such as monomer and dimer => better than filter binding
  • easier to see the complex than footprint assay
• Disadvantage: do not know where the binding site is.

Rationale

• DNA-protein complex will have a smaller mobility than that of free DNA on native gel
  • gel electrophoresis and molecular sieve
  • cage effect - difference between footprint and EMSA
• Data interpretation
  • nonspecific binding
  • smearing between DNA and complex bands

Methods (protocol)

• Lable DNA
• Mix DNA and protein to form complex
• Add nonspecific competitor
• Separate complex with free probe on a native gel