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View additional product information for LightShift™ EMSA Optimization and Control Kit - FAQs (20148X)
31 product FAQs found
NativePAGE凝胶已被成功用于EMSA，对2种纯化蛋白质间的相互作用进行分析。但是，我们尚未测试将NativePAGE凝胶用于EMSA以分析核酸（DNA或RNA）与蛋白质或蛋白质复合物间的相互作用。
The LightShift Chemiluminescent EMSA Kit is composed of two sets of components that require different storage temperatures. One component set consists of the chemiluminescent substrates and various buffers that are stored at 4°C. The other component set consists of the control DNAs and various optimization reagents that are stored at -20°C. The EBNA extract must be maintained at -20°C or it will lose activity (proteins will degrade). Short-term storage (overnight) of the other kit components at temperatures ranging from room temperature to -20°C will not adversely affect kit performance.
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This has not been tested but may be possible. A better alternative is to perform a DNA binding protein pull-down assay using a probe. The following journal article is a good example of how the LightShift Chemiluminescent EMSA Kit and pull-down assays were used to detect a transcription factor bound to a DNA probe: Ragione, A.L., et al. (2003), J. Biol. Chem. 278(26):23360-8.
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The amount of protein extract needed for a binding reaction depends on how much active DNA binding protein is in the sample. The LightShift Kit is sensitive and will easily detect 5 fmol of active protein bound to 5 fmol of biotinylated probe. If the protein being studied is abundant, 0.25 µg of a cell lysate may be sufficient for each binding reaction. However, if the protein of interest is rare, 10 µg or more of cell lysate may be needed. Using a large excess of protein extract may lead to high background signal and non-specific bands.
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A supershift assay is a method for positively identifying a protein:DNA interaction on an EMSA. An antibody (typically 1 µg) is added to the binding reaction. During electrophoresis, the antibody:protein:DNA complex migrates slowly, causing a “supershift” compared to the “shift” caused by a protein:DNA complex. Not all antibodies will cause a supershift. Some antibodies do not bind to proteins once they are bound to DNA. Some antibodies can prevent protein:DNA interactions but can still be used to confirm the identity of a protein that causes a shift in the absence of the antibody.
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Yes, the LightShift Chemiluminescent EMSA Kit can be used to detect supershifts. However, not all antibodies will work for supershift assays. Some antibodies will prevent protein:DNA interactions. In addition, the order in which the components of the binding reaction are assembled may affect the results of a supershift assay. Generally, 1 µg antibody is added as the last component in the binding reaction. For examples of how the LightShift Chemiluminescent EMSA Kit was used to detect supershifts, see the following:
Adimoolam, S. and Ford, J.M. (2002). PNAS. 99(20):12985-90
Magid, R., et al. (2003). J. Biol. Chem. 278(35):32994-9
Ragione, F.D., et al. (2003). J. Biol. Chem. 278(26):23360-8
Rinaldi, A.L., et al. (2002). Cancer Research. 62(19):5451-6
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As a control, unlabeled DNA of the same sequence as the biotin-labeled DNA is added in excess to compete with the biotin-labeled DNA. This results in a decrease or elimination of the “shift”, verifying that the protein-DNA interaction was sequence specific.
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The binding conditions for each experiment must be optimized empirically for the proteins tested. Journal articles are an excellent source for information on how to set up EMSA binding reactions. If an article describes an EMSA for your protein of interest but used 32P labeled probes, repeat the binding conditions with biotinylated probe in place of the 32P labeled probe. Although reaction components are provided with the LightShift Chemiluminescent EMSA Kit, these reagents may not be suitable for individual applications and may be adjusted as needed.
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The shift may be caused by non-specific binding or a DNA binding protein different than the one being tested.
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Non-specific bands may be reduced or eliminated by optimizing the binding reactions. Strong non-specific bands can often be mistaken for a positive result but will not be blocked by the addition of cold competitor probe. The following are examples of changes that MAY help reduce non-specific bands:
• Reduce the amount of protein extract in the binding reaction.
• Increase the amount of non-specific competitor DNA [i.e., poly(dI-dC)·poly(dI-dC)].
• Use a different non-specific competitor DNA [i.e., sonicated salmon sperm DNA or poly(dA-dT)·poly(dA-dT)].
• Preincubate the protein extract with non-specific competitor DNA before adding the biotinylated probe.
• Shorten or redesign the probe used in the experiment.
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Multiple shifts in an EMSA are most likely caused by the DNA binding to multiple forms of the protein such as a monomer vs. a dimer, or perhaps from multiple factors binding to the same site or to multiple binding sites within the target DNA (especially common with longer duplexes).
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The LightShift Chemiluminescent EMSA Kit is used to detect a biotinylated probe in an EMSA. The binding conditions for each experiment must be optimized for the proteins tested. If an experiment was working when 32P labeled probes were used, then use the same binding conditions from those previous experiments and only substitute the biotinylated probe in place of the 32P labeled probe. While commonly used reaction components are provided with the LightShift Chemiluminescent EMSA Kit, these reagents may not be applicable in all situations.
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The reaction may need to be optimized. System optimization can be achieved by adding various components supplied with the kit such as KCl, glycerol, MgCl2 and/or detergents and determining their effects on the shift. Comparing the methods used in journal articles that successfully performed EMSA experiments with the proteins you are testing may help with optimization.
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The magnitude of mobility shift will vary in different systems and depends upon the abundance and activity of the target protein. Using the control provided with the kit, approximately 50% of the biotin-labeled DNA should be shifted.
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Positively charged nylon membrane must be used (e.g., Biodyne B Membrane, Cat. No. 77016).
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Transfer to positively charged nylon membrane can be performed using an electrophoretic transfer apparatus.
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When possible, use a gel system that was used successfully by other researchers to detect your protein of interest. Commercially available DNA retardation gels often work well for EMSA applications. Both the gel type and running buffer composition can influence how well an EMSA works. The following journal article provides an overview of some considerations often overlooked by researchers performing gel-shift assays: Roder, K. and Schwiezer, M. (2001), Biotechnol. Appl. Biochem. 33:209-214.
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EMSAs may be performed with either polyacrylamide or agarose gels depending upon the resolution requirements of the study system. Traditionally, 4-6% non-denaturing polyacrylamide gels are used. If agarose is used, a capillary transfer may be best. Either capillary or electrical transfers can be performed with polyacrylamide gels.
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Typically, the target duplex in EMSA is 20-35 bp long. Our scientists have successfully used DNA sequences as long as 60 bp. The actual binding sequence for protein:DNA interactions is frequently 10-15 bp. Longer probes can be used if the binding sequence is unknown or if multiple regulatory regions are being studied.
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DNA labeled on the 5'- or 3'-end can be ordered directly from your oligo supplier or prepared using a biotin end-labeling kit such as the Biotin 3' End-Labeling Kit (Cat. No. 89818). Internal biotin labels may inhibit binding of the DNA binding protein and are not recommended.
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Yes. The Detection Module (Cat. No. 89880) and Optimization and Control Kit (Cat. No. 20148X) are available separately.
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The LightShift Chemiluminescent EMSA assay protocol, from making the DNA probe to visualization of the results, can be completed in approximately 5 hours.
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The LightShift Chemiluminescent EMSA Kit is as sensitive as 32P when optimized. In addition, the experiment can be completed in about 5 hours without the need for an overnight exposure often required when using 32P. As with every system, some optimization may be required. The LightShift Chemiluminescent EMSA Kit has superior detection sensitivity compared to DIG detection systems.
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Biotin end-labeled DNA containing a putative or known binding site is incubated with a nuclear extract or purified factor. This reaction is then subjected to gel electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane. Because DNA-protein complexes migrate slower than DNA alone in a native gel, a “shift” in the migration of the labeled DNA occurs. The biotin end-labeled DNA is detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate developed for the LightShift Kit. The signal is then detected with X-ray film or a high quality CCD camera.
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The LightShift Chemiluminescent EMSA Kit uses proprietary chemiluminescent detection technology to perform nonradioactive EMSA or supershift assays.
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EMSAs (also called gel shifts, band shifts, gel retardation assays, or mobility assays) have been used extensively for studying protein-DNA interactions. Because protein-DNA complexes migrate more slowly through a native polyacrylamide or agarose gel than DNA alone, individual protein-DNA complexes can be visualized as discrete bands within the gel using chemiluminescence or radioisotopic detection.
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Yes. You may use the same method that was used with the G2 Fast Blotter: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-biology-application-notes/transfer-emsa-gels-using-g2-fast-blotter.html
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Here are some suggestions to improve the results:
Speckling/spots can be caused by precipitate in the HRP conjugate or by air bubbles. Precipitate in the HRP conjugate can be removed by filtering the conjugate through a 0.2 µm filter or by centrifugation for 1 minute at maximum speed while air bubbles between the gel and the membrane should be removed before transfer.
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Here are possible causes and solutions:
- Particulate in Blocking Buffer or Wash Buffer: Gently warm until no particulate remains.
- Contaminants in the TBE: Use high-quality reagents or filter TBE through a 0.2 µm filter before use.
- The transfer unit or sponges used were dirty: Use clean equipment and sponges that were not previously used for western blotting.
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NativePAGE gels have been successfully used to perform EMSA showing protein-protein interactions between two purified proteins, but we have not tested NativePAGE gels for EMSA analysis of interactions between nucleic acids (either DNA or RNA) and a protein or protein complex.
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