The Thermo Scientific LightShift Chemiluminescent EMSA Kit is an extraordinarily robust and sensitive system for performing electrophoretic mobility shift assays (EMSA) to identify and characterize protein-DNA binding interactions. The kit includes reagents for setting up and customizing DNA binding reactions, a control set of DNA and protein extract to test the kit system, stabilized streptavidin-HRP conjugate to probe for the biotinlabeled DNA target, and an exceptionally sensitive chemiluminescent substrate module for detection.
Features of the LightShift Chemiluminescent EMSA Kit:
• Excellent for detecting low-abundance proteins in nuclear extracts • Sensitivity that surpasses radioactive and digoxigenin methods • Compatible with previously established binding conditions for popular DNA-protein interactions • Includes EBNA control system to help new users develop a working assay and understand the methods used to confirm binding interaction specificity
The principle for LightShift EMSA Detection is similar to a Western blot. Biotin end-labeled duplex DNA is incubated with a nuclear extract or purified factor and electrophoresed on a native gel. The DNA is then rapidly (30 minutes) transferred to a positive nylon membrane, UV crosslinked, probed with streptavidin-HRP conjugate and incubated with the substrate. The protocol from labeling to results can be accomplished in a single day.
The interaction of proteins with DNA is central to the control of many cellular processes including DNA replication, recombination and repair, transcription, and viral assembly. One technique that is central to studying gene regulation and determining protein:DNA interactions is the electrophoretic mobility shift assay (EMSA).
The EMSA technique is based on the observation that protein:DNA complexes migrate more slowly than free DNA molecules when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis. Because the rate of DNA migration is shifted or retarded upon protein binding, the assay is also referred to as a gel shift or gel retardation assay. Until conception of the EMSA protein:DNA interactions were studied primarily by nitrocellulose filter-binding assays.
All that is needed to perform the assay is purified DNA target that has been end-labeled with biotin, the protein extract to be tested, nylon membrane and basic electrophoresis equipment. DNA targets can be synthesized with 5' or 3' biotin labels or they can be labeled after synthesis using the Thermo Scientific Biotin 3' End DNA Labeling Kit (Product No. 89818). Nuclear, cytosolic or whole cell protein extracts can be obtained by a variety of methods, including the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Product No. 78833).