LightShift™ Chemiluminescent EMSA Kit

Catalog number: 20148

Thermo Scientific™  Related applications: Nucleic Acid Gel Electrophoresis & Blotting

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Description

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).

More Product Data
Transfer EMSA gels using the Pierce G2 Fast Blotter

Related Products
LightShift™ EMSA Optimization and Control Kit
LightShift™ Poly (dI-dC)
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Technique: Enhanced Chemiluminescence, Gel Shift, Membrane Blot
Assay Type: EMSA Assay
Product Size: 100 reactions
Detection Method: Chemiluminescent
For Use With (Equipment): X-Ray Film, myECL™ Imager

Contents & storage

Sufficient For: 100 binding reactions and detection on 800 cm2 of membrane (10 mini-gel blots)
• 10X Binding Buffer, 1 mL (store at -20°C)
• Biotin-EBNA Control DNA, 50 µL (store at -20°C)
• Unlabled EBNA DNA, 50 µL (store at -20°C)
• EBNA Extract, 125 µL (store at -20°C)
• Poly(dl·dC), 125 µL (store at -20°C)
• 50% Glycerol, 500 µL (store at -20°C)
• 1% NP-40, 500 µL (store at -20°C)
• 1 M KCl, 1 mL (store at -20°C)
• 100mM MgCl2, 500 µL (store at -20°C)
• 200mM EDTA, pH 8.0, 500 µL (store at -20°C)
• 5X Loading Buffer, 1 mL (store at -20°C)
• Stabilized Streptavidin-HRP, 1.5 mL (store at 4°C)
• Luminol/Enhancer Solution, 80 mL (store at 4°C)
• Stable Peroxidase Solution, 80 mL (store at 4°C)
• Blocking Buffer, 500 mL (store at 4°C)
• 4X Wash Buffer, 500 mL (store at 4°C)
• Substrate Equilibration Buffer, 500 mL (store at 4°C)

Documents

Manuals & protocols