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LightShift Chemiluminescent EMSA Kit


For gel-shift assays without radioisotopes or digoxigenin.

LightShift Chemiluminescent EMSA Kit

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.

Highlights:

  • 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

Product Details:

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.

Summary of chemiluminescent nonradioactive EMSA protocol for gel shift assays
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).

Control system for EMSA detection method validation
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 50ng/µL poly(dI•dC), 10% glycerol and 0.05% NP-40. Exposure time was 30 seconds with X-ray film.

Example application data for chemiluminescent EMSA and gel shift assays.
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-κB 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-κB.

LightShift Chemiluminescent EMSA Kit sensitivity
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 6.8µg 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. Performance of the LightShift Chemiluminescent EMSA Kit vs. radioactive (32P) EMSA

 

More Product Data

References:

  1. Guadix J. A. et al. (2011) Wt1 controls retinoic acid signalling in embryonic epicardium through transcriptional activation of Raldh2. Development. 138, 1093-7.
  2. Gau B.-H. et al. (2011) FUBP3 interacts with FGF9 3' microsatellite and positively regulates FGF9 translation. Nucleic Acids Res., gkq1295.
  3. Cai Q. et al. (2011) Replication and functional genomic analyses of the breast cancer susceptibility locus at 6q25.1 Generalize its importance in women of Chinese, Japanese, and European ancestry. Cancer Res. 71, 1344-55.
  4. Yoo C. Y. et al. (2010) The Arabidopsis GTL1 transcription factor regulates water use efficiency and drought tolerance by modulating stomatal density via transrepression of SDD1. Plant Cell. 22, 4128-41.
  5. Sultana H. et al. (2010) Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks. J. Exp. Med. 207, 1727-43.
  6. Husain M. et al. (2010) Redox sensor SsrB Cys203 enhances Salmonella fitness against nitric oxide generated in the host immune response to oral infection. PNAS. 107, 14396-401.

Related Resources:

LightShift Chemiluminescent EMSA FAQ
Protein-nucleic acid interactions technical guide
Protein-DNA interaction detection technical guide
Gel shift assays (EMSA) technical guide

Related Products:

Biotin 3´ End DNA Labeling Kit
NE-PER Nuclear and Cytoplasmic Protein Extraction Reagent Kit
Biodyne B Nylon Membrane for Chemiluminescent EMSA
Chemiluminescent Nucleic Acid Detection Module
LightShift Chemiluminescent RNA EMSA Kit


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
20148 LightShift Chemiluminescent EMSA Kit
Formulation: Multi-component kit with reagents for DNA-protein binding reactions and blot detection
Sufficient For: 100 binding reactions and detection on 800cm2 of membrane (10 mini-gel blots)

Kit Contents:
Optimization Kit (Part No. 20148X):
10X Binding Buffer, 1mL
Biotin-EBNA Control DNA, 50µL
Unlabled EBNA DNA, 50µL
EBNA Extract, 125µL
Poly(dl·dC), 125µL
50% Glycerol, 500µL
1% NP-40, 500µL
1 M KCl, 1mL
100mM MgCl2, 500µL
200mM EDTA, pH 8.0, 500µL
5X Loading Buffer, 1mL

Detection Module (Part No. 89880):
Stabilized Streptavidin-HRP, 1.5mL
Luminol/Enhancer Solution, 80mL
Stable Peroxidase Solution, 80mL
Blocking Buffer, 500mL
4X Wash Buffer, 500mL
Substrate Equilibration Buffer, 500mL
100-rxn kit Product Instructions for product #20148 LightShift Chemiluminescent EMSA Kit MSDS for product #20148 LightShift Chemiluminescent EMSA Kit Certificate of Analysis for product #20148 LightShift Chemiluminescent EMSA Kit $420.00

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20148X LightShift EMSA Optimization and Control Kit
Formulation: Multi-component kit with reagents for DNA-protein binding reactions
Sufficient For: 100 binding reactions

Kit Contents:
10X Binding Buffer, 1mL
Biotin-EBNA Control DNA, 50μL
Unlabeled EBNA DNA, 50μL
EBNA Extract, 125μL
Poly (dI·dC), 125μL
50% Glycerol, 500μL
1% NP-40, 500μL
1 M KCl, 1mL
100mM MgCl2, 500μL
200mM EDTA pH 8.0, 500μL
5X Loading Buffer, 1mL
100-rxn kit MSDS for product #20148X LightShift EMSA Optimization and Control Kit Certificate of Analysis for product #20148X LightShift EMSA Optimization and Control Kit $141.00

Add to cart
20148E LightShift Poly (dI-dC)
Formulation: 1μg/μL solution of poly(dI-dC)
Sufficient For: 100 binding reactions
125µL MSDS for product #20148E LightShift Poly (dI-dC) $74.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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