EMSA was performed using the LightShift Chemiluminescent kit from Thermo Scientific (Rockford, IL, USA) following the manufacturer’s instructions. Briefly, 5 μg of nuclear extract was incubated for 20 min on ice with biotin labeled oligos containing NF-κB binding sites (−218 bp position) of the TGF-α promoter. The DNA-protein complexes were resolved in 8% non-denaturing DNA polyacrylamide gels and transferred to nylon membrane. The complexes were detected with the Chemiluminescent Nucleic Acid Detection Module from Thermo Scientific. The primers pairs used were: (TGF-α −218) 5’-CTG TGC CCT CAG GGG GGC ACC CCC ATC GG −3’ and 5’-CCG ATG GGG GTG CCC CCC TGA GGG CAC AG −3’. These oligos were HPLC purified and biotin labeled from Operon Technologies. To confirm the specificity of the binding, the unlabeled control oligos were used in excess during the reaction.
Lightshift chemiluminescent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LightShift Chemiluminescent kit is a lab equipment product designed for the detection and analysis of protein-DNA interactions. It utilizes chemiluminescent technology to visualize and quantify these interactions.
Automatically generated - may contain errors
6 protocols using lightshift chemiluminescent kit
1
Electrophoretic Mobility Shift Assay of NF-κB
2
Purification and EMSA of ERF114 Protein
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The coding region of the ERF114 gene was cloned into vector pET‐32. Recombinant plasmid with glutathione S‐transferases (His) tag was transformed into Escherichia coli BL21 (DE3) and then induced with 0.2 mM isopropyl‐β‐d ‐thiogalactoside (IPTG) at 20°C for 12 h. Cell pellets were collected and lysed by sonication in Tris‐HCl. His‐tagged protein was purified with His‐bind resin according to the manufacturer's instructions. Protein purification and quantification were performed using Ni‐NTA Resin (DP101; TransGen Biotech) and a BCA Protein Assay Kit (DQ111; TransGen Biotech), respectively. Next, 40‐bp probes within the indicated DNA fragment in the ERF114 promoter were labelled with biotin (Table S1 ). EMSA was conducted using a Light Shift Chemiluminescent Kit (Thermo Scientific).
3
Studying NF-κB Binding in DUOX2 Promoter
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EMSA was performed with a LightShift Chemiluminescent kit according to the manufacturer’s recommendations (Cat no: 20148, Thermo Scientific, Waltham, MA, USA). Briefly, NCI-N87 cells were treated as described in [18 (link)] with the exception that irradiation was performed b.i.d. Nuclear proteins were extracted and 1.6 μg was used with 40 fmole of 5′ biotinylated DNA probe (IDT, Coraville, IA, USA). The DNA probe was a double stranded 40 nucleotides sequence from the human DUOX2 promoter containing one NF-κB binding site:
Biotin-5′-GATGGCAGCGGTGCAGGGGAATTCCCCGGGGGAGAAGCGG-3′. Where indicated 0.5 μg of p-p65-NF-κB (Ser536) monoclonal rabbit antibody (Cat no 3033; Cell Signaling, Danvers, MA, USA) was used.
Biotin-5′-GATGGCAGCGGTGCAGGGGAATTCCCCGGGGGAGAAGCGG-3′. Where indicated 0.5 μg of p-p65-NF-κB (Ser536) monoclonal rabbit antibody (Cat no 3033; Cell Signaling, Danvers, MA, USA) was used.
4
Electrophoretic Mobility Shift Assay for REST Binding
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EMSA was performed using a LightShift Chemiluminescent kit (Thermo Scientific) according to the manufacturer's instructions. Briefly, 5 μg of nuclear extract from cells were incubated with biotin-labeled oligonucleotides containing REST consensus binding sites of the EAAT2 promoter for 20 min on ice. The DNA–protein complexes were resolved on 8% nondenaturing DNA polyacrylamide gels and transferred to nylon membranes. DNA–protein complexes were detected using a Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific). The primers pairs used for REST (EAAT2 promoter including the RE1 consensus site) were 5′-GAG GAG GGA GCG CCA GGG GCT GCT CCA GGG A-3′ and 5′-TCC CTG GAG CAG CCC CTG GCG CTC CCT CCT C-3′ for −663 and 5′-CGC AGC AAA GCA CAG GTG GCA GCG GCT GCA G-3′ and 5′-CTG CAG CCG CTG CCA CCT GTG CTT TGC TGC G-3′ for −131 site.
5
BMP4-Induced SMAD1 DNA Binding Assay
6
Expression and Purification of MYC2 Protein
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The full length of MYC2 linked with the His-tag was amplified and introduced into the pMal-c2X plasmid. The recombinant plasmid was then transformed into Escherichia coli BL21(DE3) for protein expression. Protein purification and quantification were performed using Ni-NTA Resin (DP101; TransGen Biotech) and a BCA Protein Assay Kit (DQ111; TransGen Biotech), respectively. Next, 40-bp probes within the G-box motif in the ZAT18 promoter were labeled with biotin (Table S1). The EMSA was conducted using a Light Shift Chemiluminescent Kit (Thermo Scientific, Rockford, IL, USA).
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