Electrophoretic Mobility Shift Assay, or EMSA for short, is a molecular biology technique used to analyze protein-DNA interactions. The technique was discovered by Dr. Michael Fried in 1981 and is also known as the gel mobility shift assay, band shift assay, or gel retardation assay. The general principle is that when subjected to electrophoresis, free DNA will migrate differently than a DNA-protein complex. When a protein binds to a DNA fragment it hinders the fragment’s movement through a gel. The end result is a bound DNA gel band that is shifted higher on the gel compared to the unbound DNA band.

The components:

  • DNA: DNA fragments used in the EMSA generally range from 25 to 500 base pairs. Larger DNA fragments can be obtained by cutting a plasmid with restriction enzymes. Smaller fragments can be prepared by synthesizing two complementary oligonucleotides and annealing them. The DNA can be labeled in several different ways to show its final position on the gel. The fragment may be biotin-labeled, fluorescent-labeled or, most often, labeled with the radioisotope Phosphate-32.
  • Protein: Either an isolated protein or a mixture of proteins can be analyzed. The proteins must remain in their native form in order for them to be able to bind the DNA. The protein is not labeled.
  • Nondenaturing polyacrylamide gel: The gel is nondenaturing to help keep the proteins in their native form.

The general protocol:

  1. Obtain the protein of interest. Label the DNA fragment with Phosphate-32 or a biotin or fluorescent marker.
  2. Mix the protein and DNA together and incubate to let any protein-DNA interactions occur.
  3. Stop the reaction and load the mixture onto a native polyacrylamide gel.
  4. Run the gel and dry it for better preservation. Expose the gel to film to detect the position of the radiolabeled DNA.

This assay can not only determine if there is a protein-DNA interaction but also can determine the affinity, abundance, association and dissociation constants, and binding specificity of this interaction. The EMSA protocol is commonly modified in the following ways:

  • To determine which specific DNA sequence that a certain protein can recognize and bind. Multiple experiments are performed using different DNA sequences and the same protein.
  • To determine the affinity of a protein for a certain DNA sequence. This is measured by also introducing either the unlabeled original sequence or other sequences that compete with the original sequence for binding to the protein.
  • To identify any proteins in a mixture that bind to a specific DNA sequence. Here, an antibody that binds to a certain protein is mixed with the proteins and DNA. This will create a ternary complex consisting of antibody-protein-DNA that will further hinder the DNA fragment. The complex will be represented by another band on the gel that is shifted even higher than the protein-DNA band.
  • Additionally, with some minor modifications this technique can also be used to measure protein-RNA, RNA-DNA, and RNA-RNA interactions.

EMSA is a simple, rapid, and very sensitive method for detecting protein-DNA interactions, but it may take several tries to obtain an interaction. Certain variables such as buffer composition may need to be altered to optimize the binding. To make it even easier, kits have recently been developed by various biotechnology companies to streamline the process.

However, the major problem of EMSA is that it is an in vitro technique that does not accurately represent in vivo conditions. There is a decent chance that a protein that binds to a DNA sequence in an EMSA will not bind to the same sequence in a cell. Keep in mind that a short fragment of DNA in a buffered solution is a far cry from the long and wrapped chromosomal DNA in a living cell. Other DNA binding proteins or histones may prevent the protein from accessing and binding to the DNA sequence in vivo. Other techniques such as DNA footprinting, UV cross-linking, or DNA filter assays, should also be performed to help verify that an interaction occurs in vivo.

Source: Current Protocols in Molecular Biology Volume 2, Section 12. 1998

Log in or register to write something here or to contact authors.