The kit includes reagents for setting up and customizing protein:DNA binding reactions, a control set of DNA and protein extract to test the kit system, stabilized streptavidin-HRP conjugate to probe for the biotin-labeled DNA target, and an exceptionally sensitive chemiluminescent substrate module for detection.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. All you need to perform the assay are purified DNA target that has been end-labeled with biotin, the protein extract you wish to test, nylon membrane and basic electrophoresis equipment. DNA targets may be synthesized with 5' or 3' biotin labels or they may be labeled after synthesis using the Thermo Scientific Biotin 3' End DNA Labeling Kit (Part No. 89818). Nuclear, cytosolic or whole cell protein extracts may be obtained by a variety of methods, including the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Part No. 78833).
- Includes EBNA control system to help new users develop a working assay and understand the methods used to confirm binding interaction specificity
- 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