For gel-shift assays without radioisotopes or digoxigenin.
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.
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.
Procedure summary for the Thermo Scientific LightShift EMSA Kit.
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).
EMSA results using the EBNA control system. Biotin-labeled 60 bp duplex bearing the EBNA-1 binding sequence was incubated with an extract in which the EBNA-1 protein was overexpressed. The binding buffer was supplemented with 50 ng/µl poly(dI•dC), 10% glycerol and 0.05% NP-40. Exposure time was 30 seconds with X-ray film.
Chemiluminescent EMSA of four different DNA:protein complexes. Biotin-labeled target duplexes ranged in size from 21-25 bp. The EBNA reactions were supplemented with 2.5% glycerol and 0.05% NP-40, and the AP1 reactions were supplemented with 10% glycerol. The source of the Oct-1, AP1 and NF-kB transcription factors was a HeLa nuclear extract. EBNA-1 extract is provided as a control in the kit. Unlabeled specific competitor sequences (where used) were present at a 200-fold molar excess over labeled target. X-ray film exposure times for each system ranged from 2 minutes for EBNA, Oct-1 and AP1, and 5 minutes for NF-kB.
More sensitive EMSA detection than other gel shift assay methods. Comparison of the LightShift EMSA Kit to a popular digoxigenin-based EMSA kit and a radioactive method. Serial dilutions of a labeled DNA duplex were electrophoresed on a 6% polyacrylamide gel and detected according to the manufacturer’s instructions. Comparable sensitivity (<50 attomoles) was achieved with the LightShift EMSA Kit and radioactivity (2,000 cpm 32-P/fmol), although a significantly longer exposure was required for the 32P-labeled DNA. Equivalent exposures using the two chemiluminescent kits showed that the sensitivity of the LightShift EMSA Kit was approximately eight-fold greater than that of the digoxigenin kit.
Better EMSA results in less time. Comparison of the LightShift EMSA Kit to a popular digoxigenin-based EMSA kit and a radioactive method. A 22-bp duplex containing the binding sequence for the transcription factor Oct-1 was labeled for use in either the LightShift EMSA Kit, a digoxigenin-based EMSA kit or with 32P using T4 polynucleotide kinase (40,000 cpm/reaction) for use in traditional radioactive EMSA. Binding reactions were equivalent in that 20 fmol duplex was incubated with 6.8 µg HeLa cell NE-PER Nuclear Extract (where indicated). The chemiluminescent kits were used according to the manufacturer’s instructions. For the radioactive EMSA, the gel was exposed directly to X-ray film using screens.
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