What is EMSA protocol?
Introduction. The electrophoresis mobility shift assay (EMSA) is a rapid and sensitive method to detect protein–nucleic acid interactions1,2,3,4,5,6. It is based on the observation that the electrophoretic mobility of a protein–nucleic acid complex is typically less than that of the free nucleic acid (Fig. 1).
What is gel shift assay used for?
The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems.
How does EMSA assay work?
In an EMSA, or simple ‘gel shift’, a 32P-labeled DNA fragment containing a specific DNA site is incubated with a candidate DNA-binding protein. The protein-DNA complexes are separated from free (unbound) DNA by electrophoresis through a nondenaturing polyacrylamide gel.
What does the antibody Supershift control for in the gel shift assay?
The ‘gel super shift’ assay refers to the additional increase in apparent molecular weight resulting from binding a specific antibody to the DNA binding protein prior to reaction with radioactive probe.
What is needed for a gel shift assay?
The gel shift assay consists of three key steps: (1) binding reactions, (2) electrophoresis, (3) probe detection. The order of component addition for the binding reaction is often critical. Completed binding reactions are best electrophoresed immediately to preserve potentially labile complexes for detection.
Why EMSA is done?
An electrophoretic mobility shift assay (EMSA, also known as a gel shift assay) is used to determine if a protein is able to directly interact with a short, specific sequence of DNA.
What is a Supershift assay?
definition. The EMSA supershift is a EMSA experiment carried out using a third lane loaded with the radiolabeled nucleic acid, a protein mixture and an antibody for a specific protein. If an extra retardation is observed, this is due to the formation of a larger complex including the antibody.
What is a ChIP assay?
Chromatin immunoprecipitation (ChIP) assays identify links between the genome and the proteome by monitoring transcription regulation through histone modification (epigenetics) or transcription factor–DNA binding interactions.
What is the gel shift assay?
The gel shift, or electrophoretic mobility shift, assay provides a simple and rapid method for detecting DNA-binding proteins. Promega has developed the Gel Shift Assay System, which contains target oligonucleotides, a control extract containing DNA-binding proteins, binding buffer and reagents for phosphorylating oligonucleotides.
What are nonspecific competitors in DNA gel shift assays?
The most common nonspecific competitors used in DNA gel shift assays are sonicated salmon sperm DNA and poly (dI•dC). These repetitive fragments, or polymers, provide an excess of nonspecific sites to adsorb proteins in crude lysates that will bind to any general DNA sequence.
What is an electrophoretic mobility shift assay?
One important technique for studying gene regulation and determining protein–DNA interactions is the electrophoretic mobility shift assay (EMSA). An advantage of studying protein–DNA interactions by an electrophoretic assay is the ability to resolve complexes of different stoichiometry or conformation.
What is EMSA (gel shift)?
Introduction to the EMSA (gel shift) technique. The EMSA technique is based on the observation that protein–DNA complexes migrate more slowly than free linear DNA fragments when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis.