Lightshift emsa optimization control kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The LightShift™ EMSA Optimization & Control Kit is a laboratory product designed to facilitate the optimization and control of Electrophoretic Mobility Shift Assay (EMSA) experiments. The kit contains various components to assist in the optimization of EMSA procedures, including positive and negative control samples, binding buffers, and other necessary reagents.

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Lab products found in correlation

Chemiluminescent nucleic acid detection module, by Thermo Fisher Scientific (3 mentions) Nylon membrane, by Cytiva (2 mentions) Mbp beads, by New England Biolabs (1 mentions) Hybond nylon membrane, by GE Healthcare (1 mentions) Trans blot semi dry transfer cell, by Bio-Rad (1 mentions) Hybond, by Cytiva (1 mentions) Nylon membrane, by Merck Group (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Emsa binding buffer, by Thermo Fisher Scientific (1 mentions) Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions) Chemidoc xrs scanner, by Bio-Rad (1 mentions) Pcold tf vector, by Takara Bio (1 mentions) Hissep ni nta agarose resin, by Yeasen (1 mentions)

Close spelling variants of this product

EMSA kit (101 citations) LightShift EMSA Optimization and Control Kit (18 citations) LightShift EMSA kit (7 citations)

8 protocols using lightshift emsa optimization control kit

SARS-CoV-2 RdRp Enzymatic Assay

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The SARS-CoV-2 RdRp complex (0, 0.5, 1.0, 1.5, 2.0, and 3.0 μM) was incubated with 3.0 nM biotin labeled self-priming RNA, 2 U/μl RNase inhibitor, and 10 mM ATP in reaction buffer (20 mM Tris, pH 8.0, 1 mM DTT, 6 mM MgCl₂, 10 mM KCl, and 0.01% Triton-X 100) for 60 min at 37°C. The reactions were stopped by 40 μl of quench buffer (94% formamide, 30 mM EDTA, prepared with DEPC-treated water). The reaction products were loaded into a 10% Urea-PAGE denatured gel, running at 120 V for 1.5 h. After electrophoretic transfer to nylon membrane (Amersham Biosciences), the labeled RNA was detected by LightShift™ EMSA Optimization & Control Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The inhibition assays of RdRp inhibitors are similar to the above for the RdRp enzymatic assays, except inhibitors were added to reactions for 30 min before the addition of 10 mM ATP.

Bai X., Sun H., Wu S., Li Y., Wang L, & Hong B. (2022). Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay. Frontiers in Immunology, 13, 844749.

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Characterizing ZJSEP3 Protein-DNA Interactions

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EMSAs were conducted using a LightShift™ EMSA Optimization & Control Kit (Thermo Prod#20148X) and a Chemiluminescent Nucleic Acid Detection Module (Thermo 89,880) in accordance with the manufacturer’s protocol. The recombinant maltose-binding protein (MBP)-ZjSEP3 protein and MBP protein were purified from E. coli BL21 using MBP beads (New England BioLabs). The DNA fragments and mutated sequences of the LHY promoter were synthesized and labeled with biotin at the 5′ DNA terminus for serving as biotin probes. Biotin-unlabeled fragments of the same sequences or mutated sequences were used as cold probes. MBP alone was used as the negative control.

Gao W., Zhang L., Wang J., Liu Z., Zhang Y., Xue C., Liu M, & Zhao J. (2021). ZjSEP3 modulates flowering time by regulating the LHY promoter. BMC Plant Biology, 21, 527.

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Electrophoretic Mobility Shift Assay for NigR

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EMSA was performed using a LightShift^™ EMSA Optimization & Control Kit (Thermo-Fisher Scientific, Waltham, MA) with biotin-labeled DNA probes and purified NigR according to the manufacturer’s instructions. Poly(dI-dC) was used in all reactions as a non-specific competitor at a concentration of 1 μg per reaction (Ajdic & Ferretti, 1998 (link); Nieto et al., 2001 (link); Chawla et al., 2010 (link)). Unlabeled specific competitor was added where indicated. Both non-specific and specific competitors were used as described previously (Ajdic & Ferretti, 1998 (link); Gaigalat et al., 2007 (link); Nentwich et al., 2009 (link)). An unrelated protein InvB (Type III secretion chaperone from Salmonella enterica; Lilic et al., 2006 (link)) was used as a negative control. The 20-μl binding reactions were incubated at room temperature for 20 min and the DNA–protein complex was separated from the free probe by electrophoresis on 4 or 5% native polyacrylamide gel in TBE buffer. The material was transferred to positively charged Hybond nylon membrane (GE Healthcare, Pittsburgh, PA) using Trans-Blot Semi Dry Transfer Cell (Bio-Rad) according to the manufacturer’s instructions. The membrane was cross-linked for 10 min using UV Crosslinker (UVP HL-200 HybriLinker) and developed using Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific) following the manufacturer’s instructions.

Vujanac M., Iyer V.S., Sengupta M, & Ajdic D. (2015). Regulation of Streptococcus mutans PTSBio by the transcriptional repressor NigR. Molecular oral microbiology, 30(4), 280-294.

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EMSA Analysis of saxF Promoter

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EMSAs were performed as described previously (Wang et al., 2018 (link)). The saxF promoter region (250 bp) was amplified by PCR using the 5′‐biotin‐labelled primers p‐saxF‐F/R (Table S2). EMSA was carried out using the LightShift EMSA Optimization & Control Kit (Thermo) as recommended by the manufacturer with some modifications. In brief, 5 ng of the biotin‐labelled probe and a series of SstF‐GST fusion protein concentrations (0 to 4 μg) were added to the reaction solution. SFN (20 μM) was included as appropriate in this assay. After incubation at 25°C for 10 min, the products were loaded onto a native 8% (wt/vol) polyacrylamide gel, electrophoresed in 0.5× TBE buffer for approximately 1.5 h at 100 V, and then transferred to a nylon membrane (Millipore). Protein–DNA complexes were visualized by VersaDoc (Bio‐Rad).

Wang B., Xu Z., Zhao Y., Wu G., Li K., Hou R., Guo B., Tang B., Zhao Y, & Liu F. (2023). SstF, a novel sulforaphane‐sensing transcription factor of Xanthomonas campestris, is required for sulforaphane tolerance and virulence. Molecular Plant Pathology, 24(5), 452-465.

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Electrophoretic Mobility Shift Assay for RdRp Binding

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To verify the binding activity of the RdRp complex, an electrophoretic mobility shift assay was performed. The RdRp complex was preincubated at 4°C overnight and then incubated with S1^*/S2 RNA primer/template in EMSA binding buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 30°C. Samples were resolved on 6% native polyacrylamide gels running in 0.5×TBE buffer at 120 V for 45 min in a 4°C room and then transferred to nylon membrane (Amersham Biosciences) running in 0.5×TBE buffer at 15 V for 40 min. The biotin-labeled RNA was detected by LightShift™ EMSA Optimization& Control Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol and imaged by the ChemiDoc™ Imaging System (Bio-Rad Laboratories).

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Transcription Factor Binding Assay

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Nuclear extracts were prepared from HEK293T cells using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific, Waltham, MA, United States). DNA oligonucleotides for each variant were synthesized with 5′-biotin labeling and HPLC purified by Genscript Biotechnology Co. (Nanjing, China; probe sequences are listed in Supplementary Table S5). Double-stranded DNA probes were prepared by combining sense and antisense oligonucleotides, heat annealing, and slow cooling. Probes and HEK293T cell nuclear extracts were then incubated by using the LightShift EMSA Optimization & Control Kit (Thermo Scientific) at 4°C for 20 min. For competition assays, unlabeled competitors at 2-fold, 5-fold or 100-fold excess oligonucleotides were added to the reaction mixture 10 min before the addition of labeled probes. After incubation, binding reactions were separated on a 6% polyacrylamide gel, transferred blots were developed using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific), and signals were visualized with the ChemiDoc XRS + scanner (BIO-RAD, Louisville, KY, United States).

Lu C., Wang H.J., Song J.Y., Wang S., Li X.Y., Huang T, & Wang H. (2022). Fine Mapping of the MAP2K5 Region Identified rs7175517 as a Causal Variant Related to BMI in China and the United Kingdom Populations. Frontiers in Genetics, 13, 838685.

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GATA-3 Protein Binding Assay

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The gel shift assay was performed using the LightShift^TM EMSA Optimization & Control Kit (#20148, Thermo Scientific) according to the manufacturer’s manual. The purified human GATA-3 protein was from OriGene. The 6% DNA retardation gels, biotin labeled G3RE1 probe (5'-TAGGAAGCTCCGATACCAATAGCCCT-3'), unlabeled G3RE1 probe, unlabeled mutant G3RE1 probe (5'-TAGGAAGCTCCCATGGCAATAGCCCT-3'), wild type G3RE2 probe (5'-TAACTACCCCAGATAAGAAGGAGTGA-3'), unlabeled wild type G3RE2 probe, and unlabeled mutant G3RE2 probe (5'-TAACTACCCCAGAGTTGAAGGAGTGA-3') were synthesized by Invitrogen.

Kao S.Y, & Stankovic K.M. (2015). Transactivation of human osteoprotegerin promoter by GATA-3. Scientific Reports, 5, 12479.

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Purification and EMSA of AaMYB15

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Full-length coding sequence of AaMYB15 was cloned and inserted into pCold -TF vector (Takara, Japan) for protein purification. Empty pCold -TF vector was used as negative control. The plasmids were transferred into E. coli strain Rosetta (DE3) (TransGen Biotech, China). HisSep Ni-NTA Agarose Resin and 5 mL Gravity Chromatography Colimns (Yeasen, China) were used to purify fusion proteins.
EMSA was performed according to instructions of LightShiftTM EMSA Optimization& Control Kit (Thermo Fisher Scientific, China). Biotin-labeled probes of ORAM6 and ORAM6-mutated and unlabeled probes as competitor were synthesized by Sangon, China. All primers and probes related were listed in Table S1.

Wu Z., Li L., Liu H., Yan X., Ma Y., Li Y., Chen T., Wang C., Xie L., Hao X., Kayani S.L, & Tang K. (2021). AaMYB15, an R2R3-MYB TF in Artemisia annua, acts as a negative regulator of artemisinin biosynthesis. Plant Science, 308, None.

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