What does the antibody super shift control for in the gel shift assay?
If the protein of interest binds to the target DNA, the antibody will bind to that protein–DNA complex, further decreasing its mobility relative to unbound DNA in what is called a “supershift”.
What does EMSA show?
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.
What does gel shift assay do?
What is Supershift in EMSA?
emsa supershift. 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 are the steps in the gel shift assay?
Overview of the gel shift assay method. 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.
What is the biotin label used for?
This biotin label allows for detection with avadin proteins. Use of this kit results in the 3’ end labeling of RNA probes. A nonspecific competitor is any irrelevant, unlabeled nucleic acid used as a blocking/quenching agent in the binding reaction to minimize the binding of nonspecific proteins to the labeled target DNA.
What is gel electrophoresis mobility shift assay?
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.
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.