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The Gelshift Chemiluminescent EMSA Kit provides a simple, non-radioactive assay to identify protein-DNA binding with proven reagents. In this electrophoretic mobility shift assay (EMSA), cell extracts or purified factors are incubated with biotin end-labeled probe containing the consensus binding site of interest. Samples in which the protein of interest bound the target DNA will migrate slower than DNA alone resulting in a "shift" of the labeled DNA band. For complete details, click the EMSA Method tab below.
Gelshift Chemiluminescent EMSA Kit
The Gelshift Chemiluminescent EMSA Kit includes all the reagents needed to study protein-DNA binding for your sample system. The kit also includes biotin-labeled and unlabeled control DNA and control nuclear extract. Click the Gelshift tab below for data and more information; kit manuals can be downloaded under the Documents tab.
| Name | Format | Cat. No. | Price | |
|---|---|---|---|---|
| Gelshift™ Chemiluminescent EMSA | 100 rxns | 37341 | $395 |
 |
Gelshift or Electrophoretic Mobility Shift (EMSA) Info
Electrophoretic mobility shift assays (EMSA), also known as gel shifts, gel retardation assays or mobility assays can be used to study DNA-protein interactions1-3. The principle behind EMSA relies on the fact that DNA-protein complexes migrate slower than DNA alone in a native polyacrylamide or agarose gel. This difference in electrophoretic separation of DNA-protein complexes can be visualized as a "shift" in migration of the labeled DNA band. This enables researches to screen different nuclear extracts or gene promoters for specific transcription factor DNA binding activity.
Better sensitivity
The non-radioactive format of the Gelshift Chemiluminescent EMSA Kit does not sacrifice sensitivity when compared to 32P or digoxigenin methods.
Figure 1: Gelshift Chemiluminescent EMSA Kit provides better sensitivity.
HeLa nuclear extract (6.8 µg) was incubated with 20 fmol Oct-1 duplex DNA labeled for use in either the Gelshift Chemiluminescent EMSA kit, a digoxigenin-based EMSA or with 32P using T4 polynucleotide kinase (40,000 cpm/reaction) for use in a radioactive EMSA. The kits were used according to the manufacturer's instructions. The radioactive EMSA was exposed directly to X-ray film using screens.
Figure 2: Obtain sensitive results for the DNA-protein complex of interest.
The Gelshift Chemiluminescent EMSA Kit was used to obtain sensitive gel shift results for four different DNA-protein complexes. Biotin-labeled target duplexes were incubated with HeLa nuclear extract (Oct-1, AP1 and NFκB) or the provided Control Nuclear Extract from the kit. Unlabeled competitor sequences were used at 200-fold molar excess over labeled DNA. The Control reactions from the Epstein-Barr Nuclear Antigen (EBNA) system were supplemented with 2.5% glycerol and 0.05% NP-40, while the AP1 reactions were supplemented with 10% glycerol. X-ray film exposure time varied from 2 minutes for the EBNA Control, Oct-1 and AP1 to 5 minutes for NFκB.
References
1. Fried, M. and Crothers, D.M. (1981) Nucleic Acids Res. 9: 6505-6525.
2. Revzin, A. (1989) BioTechniques 7: 346-354.
3. Hendrickson, W. (1985) BioTechniques 3: 198-207.
Gelshift Chemiluminescent EMSA Method
The Gelshift Chemiluminescent EMSA Kit provides a non-radioactive method to detect DNA-protein interactions. In this method, cell extracts or purified factor are incubated with a biotin 3' or 5' end-labeled DNA probe containing the consensus binding site of interest. Samples are then resolved by electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane. The biotin end-labeled DNA probe is detected using streptavidin conjugated to horseradish peroxidase (HRP) and a chemiluminescent substrate. Samples in which the protein of interest bound the target DNA will migrate slower than DNA alone resulting in a "shift" of the labeled DNA band.
Example EMSA Binding Reactions
- Biotin-labeled DNA = no shift in the absence of cell extract or purified factor
- Biotin-labeled DNA + Extract = DNA shift due to the binding of protein to the labled DNA probe
- Biotin-labeled DNA + Extract + Excess Unlabeled DNA = no shift as the excess of unlabeled DNA competes for binding of the target protein in the extract; this reaction verifies the specificity of the protein-DNA interaction
Gelshift Chemiluminescent EMSA Advantages
- Non-radioactive assay
- Better sensitivity than radioactive or digoxigenin methods
- Fast procedure can be completed in 5 hours
- Additional reagents supplied to help optimize conditions for your sample system
- Includes control DNA and extract to help new users understand the methods as they optimize conditions for their sample systems
Contents & Storage
The Gelshift Chemiluminescent EMSA Kits contain 10X Binding Buffer, Biotin Control DNA, Unlabeled Control DNA, Control Nuclear Extract, Poly d(I-C), 50% Glycerol, 1% NP-40, 1 M KCl, 100 mM MgCl2, 200 mM EDTA pH 8.0 and 5X Loading Buffer, which are all stored at -20°C. Additionally, the kit includes Streptavidin HRP-conjugate, Chemiluminescent Reagent, Reaction Buffer, Blocking Buffer, 4X Wash Buffer and Substrate Equilibration Buffer which should be stored at 4°C.
