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EMSA gel shift assay

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More than 424 EMSA kits now available covering most widely studied transcription factors.

  • Easy—identify DNA-binding proteins
  • Economical—all-in-one system!
  • Highly sensitive—HRP-based detection system
  • Safe—no radioactivity required

Affymetrix EMSA (Electrophoretic Mobility Shift Assay) Kits are useful tools for identifying proteins that interact with DNA. This rapid technique is based on the different electrophoretic mobilities of free DNA and DNA/protein complexes in native (non-denaturing) polyacrylamide gels. The procedure is simple:

  1. Incubate the biotin-labeled DNA probe with your protein(s) of interest (purified or crude extract).
  2. Separate the mixture on a non-denaturing polyacrylamide gel.
  3. Visualize shifted bands that correspond to the protein/DNA complexes.

Unlike other comercially available EMSA kits, Affymetrix' Kits include specific transcription factor probes. We currently offer 150 kits, which correspond to the transcription factors included on the Protein/DNA Arrays I and II. Additionally, we offer a combination kit, which includes any three probe sets. Probe sets can also be purchased separately.

emsa1.jpg

NFkB EMSA Kit.
Lane 1: Labeled EMSA Probe only with no sample.
Lane 2: Labeled EMSA Probe with untreated sample.
Lane 3: Labeled EMSA Probe with treated sample.
Lane 4: Treated sample with cold and labeled EMSA probes.

To confirm whether the DNA binding activity of a particular protein is specific, you simply add an excess of cold, unlabeled DNA probe to the mixture of probe and nuclear extract. The cold probe competes with the labeled DNA probe for binding to the protein. As a result, the band's intensity is reduced or eliminated.

How it works

Affymetrix Electrophoretic-Mobility Shift Assay (EMSA) Kits are useful tools for identifying proteins that interact with DNA. This rapid technique is based on the separation of free DNA from protein/DNA complexes due to the differences in their electrophoretic mobility in native (non-denaturing) polyacrylamide gels. When a protein binds specifically to a labeled dsDNA sequence, it migrates slower than non-bound dsDNA in a polyacrylamide gel, thus resulting in discrete bands corresponding to the individual protein/DNA complex. A typical EMSA experiment is performed by incubating a biotin-labeled transcription factor (TF) Probe with treated and untreated nuclear extracts. The protein/DNA complexes are separated on a non-denaturing polyacrylamide gel. The gel is transferred to a nylon membrane and detected using strepatvidin-HRP and a chemiluminescent substrate. The shifted bands corresponding to the protein/DNA complexes are visualized relative to the unbound dsDNA. The bands are visualized after exposure to film or chemiluminescent-imaging system.

emsa1.jpg

 NFkB EMSA Kit.
Lane 1: Labeled EMSA Probe only with no sample.
Lane 2: Labeled EMSA Probe with untreated sample.
Lane 3: Labeled EMSA Probe with treated sample.
Lane 4: Treated sample with cold and labeled EMSA probes.

To confirm whether the DNA binding activity of a particular protein is specific, you simply add an excess of cold, unlabeled DNA probe to the mixture of probe and nuclear extract. The cold probe competes with the labeled DNA probe for binding to the protein. As a result, the band's intensity is reduced or eliminated.

For more specific binding, you may wish to add unlabeled specific dsDNA probe (cold probe; provided) to the protein/DNA reaction mixture which competes with the labeled dsDNA probe (biotin-labeled probe) for binding to the protein. This causes the labeled probe to migrate to the bottom of the gel and reduces the intensity of the shifted band.

Affymetrix EMSA Kits are suitable for validation of results obtained using Procarta Transcription Factor Plex Assay, Protein/DNA Arrays (Cat. #s MA1010 to MA1015) or to validate binding activity of a specific transcription factor (protein) to DNA. Enough reagents are provided to perform 25 binding reactions. Affymetrix' TF EMSA Kits measure the activity of specific transcription factors in nuclear extracts. The assay is highly specific, precise, and requires a minimum of 5 ug of protein/well. Affymetrix currently offers more than 400 EMSA assays.