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

Procedure summary for the Thermo Scientific LightShift EMSA Kit.

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 approximay 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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Related Resources: Tech Tip: Modify and label oligonucleotide 5´ phosphate groups Tech Tip: Anneal complementary pairs of oligonucleotides LightShift Chemiluminescent EMSA FAQ 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 Sufficient components for 100 binding reactions and detection reagents for ~800 cm2 of membrane. Kit Contents: LightShift EMSA Optimization and Control Kit （20148X, available separay） 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 100 mM MgCl2, 500µL 200 mM EDTA pH 8.0, 500µL 5X Loading Buffer, 1mL Chemiluminescent Nucleic Acid Detection Module （89880, available separay） Stabilized Streptavidin-Horseradish Peroxidase Conjugate, 1.5mL Luminol/Enhancer Solution, 80mL Stable Peroxide Solution, 80mL Blocking Buffer, 500mL 4X Wash Buffer, 500mL Substrate Equilibration Buffer, 500mL Kit Local contact 20148X LightShift EMSA Optimization and Control Kit 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 100 mM MgCl2, 500µL 200 mM EDTA pH 8.0, 500µL 5X Loading Buffer, 1mL Kit Local contact 20148E LightShift Poly （dI-dC） 1µg/µL solution 125 µL Local contact