Health & Research

The Electrophoretic Mobility Shift Assay (EMSA)

The electrophoretic mobility shift assay (EMSA), also known as gel retardation or band shift assay, is a rapid and sensitive means for detecting sequence-specific DNA-binding proteins. This procedure can determine if a protein or a mixture of protein is capable of binding to a particular DNA or RNA sequence. Several nuclear mechanisms involve specific DNA–protein interactions. The electrophoretic mobility shift assay (EMSA, also known as the gel mobility shift or gel retardation assay), first described almost two decades ago provides a simple, efficient and widely used method to study such interactions.

DNA protein interaction assays

  • Electrophoretic mobility shift assay (EMSA)
  • DNase 1 Foot printing
  • Chromatin Immunoprecipitation ( Chip)

What is EMSA

  • Electrophoretic mobility shift assay (EMSA).
  • It is a regularly used system to detect protein-nucleic acid interactions.

What is mobility shift assay

A mobility shift assay is electrophoretic separation of a protein–DNA or protein–RNA mixture on a polyacrylamide or agarose gel for a short period (about 1.5-2 hr)

What is the aim of EMSA

It was originally developed with the aim of quantifying interactions between DNA and proteins. This assay can be used to determine, in both a qualitative and quantitative manner, if a particular transcription factor is present within the nuclei of the cells or tissue of interest or to identify an unknown DNA binding protein which may control the expression of your gene of interest.

Importance of DNA–Protein interaction

Interactions between proteins and nucleic acids mediate a wide range of processes within a cell. These specific interactions are crucial for control of DNA replication, regulation of transcription, and translation.

Principle of the Method

Electrophoretic mobility shift assay (EMSA) is based on the simple rationale that proteins of differing size, molecular weight, and charge will have different electrophoretic mobilities in a non-denaturing gel matrix. In the case of a DNA–protein complex, the presence of a given DNA-binding protein will cause the DNA to migrate in a characteristic manner, usually more slowly than the free DNA, and will thus cause a change or shift in the DNA mobility visible upon detection. A univalent protein, P, binding to a unique site on a DNA molecule, D, will yield a complex, PD, in equilibrium with the free components:                                     

                                                                                      Ka
                                                             P + D                         PD

Kd

where  ka is the rate of association and kd is the rate of dissociation. In the case of a strong interaction between protein and DNA, with ka > kd, two distinct bands are observed, corresponding to the complex PD and to the free DNA. In contrast, a weak DNA–protein interaction, with ka < kd, should produce a fainter band corresponding to the complex PD.

Procedure of EMSA

Overview of the Procedure

Several components are required for EMSA and may influence the outcome of the procedure.

Nuclear Extract

The choice of protein extract is governed by the objective of the study. Whole-cell or nuclear extracts are very useful in analyzing the regulatory elements of a DNA fragment such as a gene promoter. Otherwise purified protein and recombinant proteins are also used to check the protein competition and protein interaction.

DNA Probe

Cloned DNA fragments of 20-200 bp in length or synthetic oligonucleotides of 20–70 nucleotides work very well in EMSA. Although larger DNA fragments usually encompass more extensive regulatory sequences, oligonucleotides will generally contain fewer protein binding sites and thereby yield more specific information. The detection of DNA–protein complexes is usually achieved by labeling of DNA probe, and this is performed using a [32P]-labeled deoxynucleotide. However, other, less hazardous methods are available, including labeling with 33P, with biotin.

Gel Matrix

Acrylamide gels combine high resolving power with broad size-separation range and provide the most widely used matrix. Because of their larger pore size, agarose gels are sometimes used, either alone or in combination with acrylamide, to study larger DNA fragments or multiprotein complexes. Gel concentration is also important in EMSA, however although lower concentration will generally allow the resolution of larger complexes.

Buffer

Different low-ionic-strength buffers can be used in EMSA and can include cofactors such as Mg2+ or cAMP, which may be necessary for some DNA–protein interactions.

Nonspecific Competitors

To ensure specificity of the DNA–protein interaction, a variety of nonspecific competitors may be used. This is particularly important when using crude protein extracts which contain nonspecific DNA-binding proteins. To avoid nonspecific binding activities interfering with the EMSA, an excess of a nonspecific DNA such as salmon sperm DNA, calf thymus DNA or synthetic DNAs such as poly (dI:dC) is used.

Critical Parameters

In theory, EMSA is a very simple and rapid assay.  However, clean, successful gel shifts can require the optimization of a number of parameters which will influence the ability of many transcription factors to bind to their cognate DNA sequences. Parameters include percentage of acrylamide used (we typically use 4 -6% acrylamide) and buffer composition.  Most commonly Tris-acetate (TAE) or Tris-borate (TBE) buffer are used.  These buffers help to resolve the protein DNA complex from the free DNA. The choice of specific DNA probe also must be considered.  Fragments of DNA ranging in size from 20 to 200 base pairs can successfully be used in this assay.  It is important to remember that the longer the fragment of DNA that you are working with, the more likely you will be to observe multiple DNA/protein interactions. In addition, longer probe lengths make distinction of shifted complexes from unreacted probe more difficult.

Detection

For visualization purposes, the nucleic acid fragment is usually labelled with a radioactivefluorescent or biotin label. Standard ethidium bromide staining is less sensitive than these methods and can lack the sensitivity to detect the nucleic acid if small amounts of nucleic acid or single-stranded nucleic acid(s) are used in these experiments.

Advantages

  • EMSA is a basic, easy to perform, and robust method able to accommodate a wide range of conditions.
  • EMSA is a sensitive method, small sample volumes (20 μL or less).
  • Used with a wide range of nucleic acid sizes and structures as well as a wide range of proteins.
  • It is possible to use both crude protein extracts and purified recombinant proteins.

Limitations

  • Dissociation of bands can occur during electrophoresis.
  • Does not provide a straightforward measure of the weights or entities of the proteins as mobility in gels is influenced by several other factors.
  • Does not directly provide information on the nucleic acid sequence the proteins are bound to.
  • The time required to mix the binding reaction and for the electrophoretic migration to occur before the mix enters the gel.

Application:

  • Detection of DNA-binding factors/proteins
  • Analysis of DNA sequences (e. g. promoter or
    enhancer regions) for their potential to bind
    specifically to proteins/nuclear extracts
  • Analysis of cellular extracts for the presence
    of certain DNA-binding proteins (e.g a transcription
    factor with a known recognition sequence)

References 

  1. Fried,M. and Crothers,D.M. (1981). Equilibria and kinetics of lac repressor-operator

interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505-6525.

  1. Garner,M.M. and Revzin,A. (1981). A gel electrophoresis method for quantifying the

binding of proteins to specific DNA regions: Application to components of the

Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047-3060.

  1. Smith, M. F., Jr., Carl, V. S., Lodie, T. A., and Fenton, M. J. Secretory interleukin-1

receptor antagonist gene expression requires both a PU.1 and a novel composite NF

κB/PU.1/GA-binding protein binding site. Journal of Biological Chemistry 273[37],

24272-24279.

  1. Dignam,J.D., Lebovitz,R., and Roeder,R.G. (1983). Accurate transcription initiation by

RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids

Res. 11, 1475-1489

  1. Maniatis,T., Fritsch,E.F., and Sambrook,J. (1982). Spun Column procedure. In Molecular

Cloning: A Laboratory Manual, T.Maniatis, E.F.Fritsch, and J.Sambrook, eds. (Cold

Spring Harbor, NY: Cold Spring Harbor Press), pp. 466-467.

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