Electrophoretic Mobility-Shift Assay (EMSA) Kit, with SYBR™ Green & SYPRO™ Ruby EMSA stains
Invitrogen™
Electrophoretic Mobility-Shift Assay (EMSA) Kit, with SYBR™ Green & SYPRO™ Ruby EMSA stains
This fluorescence-based Electrophoretic Mobility Shift Assay (EMSA) Kit provides a fast, easy and quantitative method to detect both nucleic acidRead more
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Catalog Number
Quantity
E33075
1 kit
Catalog number E33075
Price (USD)
152.00
Each
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Quantity:
1 kit
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Price (USD)
152.00
Each
Add to cart
This fluorescence-based Electrophoretic Mobility Shift Assay (EMSA) Kit provides a fast, easy and quantitative method to detect both nucleic acid and protein in the same gel. This kit uses two fluorescent dyes for detection - SYBR™ Green EMSA nucleic acid gel stain for RNA or DNA detection and SYPRO™ Ruby EMSA protein gel stain for protein detection. The nucleic acids and proteins are stained sequentially in the gel after electrophoresis, so there is no possibility that labeling will interfere with the protein binding being assayed. Staining only takes about 20 minutes for nucleic acid, and then about 4 hours for the subsequent protein staining.
Store in freezer (-5 to -30°C) and protect from light.
Frequently asked questions (FAQs)
What is a "supershift"?
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.
What is an electrophoretic mobility shift assay (EMSA)?
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.
What is a shift assay? What is a supershift assay?
A shift assay is a DNA-binding assay using nondenaturing PAGE. It provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-binding proteins. Proteins bind specifically to an end-labeled DNA fragment corresponding to the individual protein-DNA complexes. You can use the assay to test binding of purified proteins or of uncharacterized factors in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA-binding proteins.
A supershift assay is a variation of the mobility shift DNA-binding assay that uses antibodies to identify proteins present in the protein-DNA complex.Addition of a specific antibody to a binding reaction can have one of several effects. If the protein recognized by the antibody is not involved in complex formation, addition of the antibody should have no effect. If the protein that forms the complex is recognized by the antibody, the antibody can either block complex formation or it can form an antibody-protein-DNA ternary complex and thereby specifically result in a further reduction in the mobility of the protein-DNA complex (a supershift). Results may be different depending upon whether the antibody is added before or after the protein binds DNA (particularly if there are epitopes on the DNA binding surface of the protein).
What is the composition of the 5X Binding Buffer included within Electrophoretic Mobility-Shift Assay (EMSA) Kit, with SYBR Green & SYPRO Ruby EMSA stains (Cat. No. E33075)?
Here is the composition of the 5X Binding Buffer:
- 50 mM Tris HCl, pH 7.4
- 750 mM KCl
- 0.5 mM DTT
- 0.5 mM EDTA
Note: This buffer is optimized for lac repressor binding to lac operator. It may not be optimal for other protein-nucleic acid interactions.