LightShift™ EMSA Optimization and Control Kit

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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