LightShift EMSA Optimization and Control Kit EMSA优化和对照试剂盒,LightShift EMSA Optimization and Control Kit/EMSA优化和对照试剂盒-试剂- bio-rad伯乐一级代理 |伯乐Aminex |伯乐电泳槽1658001| 伯乐ddPCR bio-rad授权代理商、biorad伯乐qpcr仪

LightShift EMSA Optimization and Control Kit EMSA优化和对照试剂盒,LightShift EMSA Optimization and Control Kit/EMSA优化和对照试剂盒

2024-10-23

LightShift EMSA Optimization and Control Kit/EMSA优化和对照试剂盒

货号：20148X

规格：100 reactions

价格：2008

产品类型：蛋白凝胶电泳

品牌：Thermo Fisher

The Thermo Scientific LightShift EMSA Optimization and Control 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 and a control set of DNA and protein extract to test the kit system.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).

特点：

• 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

数据：

图1. More sensitive EMSA detection than other gel shift assay methodsComparison 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.

图2. Procedure summary for the LightShift EMSA Kit

图3.Chemiluminescent EMSA of four different DNA:protein complexesBiotin-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.

技术参数

组成成分 • 10X Binding Buffer, 1 mL • Biotin-EBNA Control DNA, 50 µL • Unlabeled EBNA DNA, 50 µL • EBNA Extract, 125 µL • Poly (dI·dC), 125 µL • 50% Glycerol, 500 µL • 1% NP-40, 500 µL • 1 M KCl, 1 mL • 100mM MgCl2, 500 µL • 200mM EDTA pH 8.0, 500 µL • 5X Loading Buffer, 1 mL

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LightShift EMSA Optimization and Control Kit EMSA优化和对照试剂盒,LightShift EMSA Optimization and Control Kit/EMSA优化和对照试剂盒

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