PC3 cells were pretreated with 30 μM MnTE-2-PyP overnight, and then exposed to 20 Gy of radiation. The nuclear proteins were isolated with the CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich), and its concentration was quantified with using Bradford reagent (Amresco). The following oligonucleotide corresponding the sequence harboring the HRE of human PAI-1 gene was synthesized: 5′-CTGACACTGCAC GTCAGAAGGACA-3′ (the element present in the promoter region is underlined). The oligonucleotides were end-labelled using the Biotin 3′ End DNA Labelling kit (Thermo Scientific). The EMSAs were performed using the Light Shift Chemiluminescent EMSA kit (Thermo Scientific). A biotin-labelled oligonucleotide (20 fmol) was incubated with 10 μg of nuclear protein extract for 20 minutes at room temperature in binding buffer. The binding complexes were resolved using electrophoresis with a 6% DNA Retardation Gel (Invitrogen) and transferred to nylon membranes (Thermo Scientific), UV cross-linked and visualized using the Chemiluminescent Nucleic Acid Detection System (Thermo Scientific).
Biotin 3 end dna labelling kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Biotin 3' End DNA Labelling kit is a tool designed to facilitate the addition of biotin labels to the 3' end of DNA molecules. It provides the necessary reagents and protocols to perform this labelling process.
Automatically generated - may contain errors
Lab products found in correlation
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (5 mentions)
Nylon membrane, by Thermo Fisher Scientific (1 mentions)
Bradford reagent, by Avantor (1 mentions)
Dna retardation gel, by Thermo Fisher Scientific (1 mentions)
Cellytic nuclear extraction kit, by Merck Group (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Nylon membrane, by GE Healthcare (1 mentions)
Biotin labelled double stranded oligonucleotides, by Eurofins (1 mentions)
Imagej software, by Thermo Fisher Scientific (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Biodyne b, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent nucleic acid detection module, by Thermo Fisher Scientific (1 mentions)
8 protocols using biotin 3 end dna labelling kit
1
Quantifying Transcription Factor Binding
2
Recombinant WRKY50BD and WRKY70 Binding
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Expression and purification of recombinant WRKY50BD and WRKY70 from E. coli and binding reactions to biotin-labelled probes and gel electrophoresis were done according to earlier protocols [23 (link), 27 (link)]. Biotin-labelling of the probes was done using the ‘Biotin 3’ End DNA Labelling Kit’ (Thermo Fisher Scientific). Probe and competitor sequences are shown in the corresponding figures. Blotting, crosslinking of the probe to the membrane, and probe detection was done according to the manual of the ‘LightShift Chemiluminescent EMSA Kit’ with a ChemiDoc™Touch Imaging System (BioRad). For each experiment the results were confirmed independently.
3
Recombinant PbrWRKY53 Protein Purification and EMSA
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The CDS of PbrWRKY53 was amplified and inserted into the pCzn1‐His vector to generate the recombinant His‐6‐PbrWRKY53 protein. The resulting construct was expressed in Escherichia coli strain BL21 (DE3) cells, and the recombinant protein was expressed and purified using Ni‐IDA resin according to the manufacturer's instructions. EMSAs were performed using the Light Shift Chemiluminescent EMSA Kit (Pierce, IL) according to the manufacturer's protocol and as described (Wu et al., 2016 ). A 30 bp biotin‐labelled DNA probes containing either WT (P1) or mutated W‐box element (mP1), and unlabelled competitor DNA were synthesized (Shanghai Sangon Biotechnology, Shanghai, China) based on the PbrNCED1 promoter sequence and labelled using the Biotin 3′ End DNA Labelling Kit (Thermo Fisher Scientific, Waltham, MA, USA). The binding reaction was performed for 20 min at room temperature in a 20 μL reaction buffer. The DNA–protein complexes were separated on 6.5% polyacrylamide gel and electrophoretically transferred to a nylon membrane (GE Healthcare, Danderyd, Sweden), and detected following the manufacturer's instructions. This experiment was conducted three times.
4
Nuclear Extract Preparation and EMSA Analysis
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Nuclear extract from Jurkat cells was prepared using the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific, Waltham, Massachusetts, USA). Electrophoretic mobility gel shift assays (EMSAs) were performed with LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Waltham, Massachusetts, USA) using 5 μg of nuclear extract and 0.6 ng biotin-labelled double-stranded oligonucleotides (50 bp fragment—Eurofins, Wolverhampton, UK). The sequences of the synthetic single-stranded oligonucleotides used in the construction of these double-stranded oligonucleotides are listed in the online supplementary methods.
Probes were prepared using a biotin 3′ end DNA labelling kit (Thermo Scientific, Waltham, Massachusetts, USA).
Single-stranded biotinylated oligonucleotides were mixed and annealed at room temperature for 1 h. Unlabelled competitor probes were used in 100-fold excess.
EMSAs were performed according to standard protocol (Thermo Scientific).
Band intensity was quantified with ImageJ software (Bethesda, Maryland, USA).
A detailed protocol is available online (see online supplementary methods section).
Probes were prepared using a biotin 3′ end DNA labelling kit (Thermo Scientific, Waltham, Massachusetts, USA).
Single-stranded biotinylated oligonucleotides were mixed and annealed at room temperature for 1 h. Unlabelled competitor probes were used in 100-fold excess.
EMSAs were performed according to standard protocol (Thermo Scientific).
Band intensity was quantified with ImageJ software (Bethesda, Maryland, USA).
A detailed protocol is available online (see online supplementary methods section).
5
Purification and EMSA of SlPIF3 bHLH Domain
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The pET‐32a‐His‐SlPIF31261–1605 vector was generated using a truncated sequence of SlPIF3 that encoded the bHLH domain. His‐tagged SlPIF3 protein was expressed in Escherichia coli strain Rosetta (DE3) and purified using a High Affinity Ni‐NTA Resin kit (GenScript, Nanjing, China). The putative target gene promoter fragments containing the G‐box and mutated G‐box region were synthesised as biotin‐labelled oligonucleotides according to a Biotin 3′ End DNA Labelling kit (Thermo Fisher Scientific, Waltham, MA, USA). Electrophoretic mobility shift assay (EMSA) was performed using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific). The sequences are listed in Dataset S1 .
6
Oligonucleotide DNA Probe Labeling and Gel Shift Assay
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The oligonucleotides were synthesized by Sangon Biotech. Double‐stranded DNA probes were 3′ end‐labelled with biotin using a Biotin 3′ End DNA Labelling Kit (Thermo Scientific). The sequences of the probes are listed in Table S1 . Nucleoproteins were incubated with the probes and Poly (dI.dC) for 20 minutes at RT before being resolved on a 6% polyacrylamide gel in 0.5 × Tris‐borate buffer, electrotransferred onto a 0.45‐μm nylon membrane (Biodyne™ B; Thermo) at 100 V for 45 min at 4°C, and then crosslinked to the nylon membrane with an 254nm UV Lamp. Bands were detected with the National Institutes of Health (NIH) ImageJ software.
7
Characterization of Os bZIP74 Transcription Factor Binding
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The OsbZIP74 probe was created by annealing together the complementing oligonucleotides and biotinylated with the Biotin 3’‐end DNA Labelling Kit (Thermo Fisher Scientific, Waltham, MA, USA). MBP‐NTL3∆C proteins were expressed in E. coli strain BL21 and purified by amylose agarose beads. EMSA was performed using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific), according to the manufacturer’s protocols. Briefly, each 20 μl binding reaction contained 2 μl binding buffer, 0.3 μl Poly (dI‐dC), 4 μg purified protein, 50 nmol biotin‐labelled probe or certain amount of unlabelled probe as the competitor. The binding reactions were allowed to incubate at room temperature for 20 min and run on a 5% non‐denaturing polyacrylamide gel. The complex was transferred to a nylon membrane (GE). After UV light cross‐linking, the DNA on the membrane was detected with the Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher Scientific).
8
Measuring Sp1/DNA Binding in Kidney Cells
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Nuclear proteins were isolated from kidney tissue and the Sp1/DNA binding activity was measured by electrophoretic mobility shift assay (EMSA). In brief, oligonucleotides were biotin-labelled by using the Biotin 3’ End DNA labelling kit (Thermo Fisher Scientific). The sequence of the Sp1 oligonucleotide probe was 5´-CTGGGCTGGGC-3´. The EMSA was performed by using the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific).
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