Electrophoretic Mobility-Shift Assays (EMSA) or Gel shift

Electrophoretic Mobility-Shift Assays (EMSA)

 Why EMSA is important?

A Method for Analysing Protein-DNA Interactions

❖EMSA is most commonly used for qualitative assays including identification of nucleic acid-binding proteins and of the respective consensus DNA sequences.

❖Under proper conditions, however, EMSA can also be used for quantitative purposes including the determination of binding affinities and kinetics

❖EMSA is a commonly used method in the characterization of transcription factors, the most intensely studied DNA-binding proteins.

Definition and Basic Principle of EMSA

❑Techniques used to study interactions between proteins and DNA.

❑Simple, efficient and sensitive technique

❑DNA moves through the gel faster when not bound to protein

❑A reduction in electrophoretic mobility shows that a complex is formed between DNA and protein

❑It can be used to identify DNA-binding proteins present in a nuclear cell extract. For example, transcription factors.

❑In an electrophoretic mobility-shift assay (EMSA, or simply “gel shift”), a 32P labeled DNA fragment containing a specific DNA site is incubated with a cognate DNA-binding protein.

❑The protein–DNA complexes are separated from free (unbound) DNA by electrophoresis through a nondenaturing polyacrylamide gel.

❑The protein retards the mobility of the DNA fragments to which it binds.

❑Thus, the free DNA will migrate faster than the DNA–protein complex.

❑An image of the gel is used to reveal the positions of the free and bound radiolabeled DNAs.

Methods of doing EMSA

5 basic steps are in conventional EMSA protocol
1. –Preparation of purified or crude protein sample
2. –Preparation of nucleic acid
3. –Binding reactions
4. –Non-denaturing gel electrophoresis
5. –Detection of the outcome

Advantages of EMSA: 

• It is a simple method to perform but yet is robust enough to include a wide range of conditions.

• Highly sensitive method. Assays could be performed with small nucleic acid concentrations and small sample volumes.

• EMSA could also be used with a wide range of nucleic acid sizes and structures as well as a wide range of proteins.

• Finally, it is possible to use both crude protein extracts and purified recombinant proteins.

Limitations of EMSA:

 Dissociation is one of the drawbacks of EMSA. It occurs during electrophoresis thus prevents detection.
• EMSA doesn’t provide information on the nucleic acid sequence the proteins are bound to.

• Not an appropriate method for Kinetic studies.

• It does not provide a straightforward measure of the weights of the proteins as mobility is influenced by several other factors.