DNA sequences were amplified by PCR with fluorescein Cy5 labelled primers using Q5® High-Fidelity Polymerase (NEB). Genomic DNA of Prochlorococcus MED4 and the cyanophage P-SSP7 were used as templates, respectively. The respective primers are listed in the Supplementary Table S1 . All four rne segments P-, P, Pi, and Pi+ were Cy5 labeled. EMSAs were conducted using 12.5 ng of the target DNA, 1 µg of Lightshift™ Poly dI-dC (Thermo Scientific™, Waltham, MA, USA) as a competitor and a final concentration of 2 mM DTT. A 1 × reaction buffer (20 mM Tris (pH8), 60 mM KCl, 1 mM EDTA and 12% glycerol (w/v)) was used for each reaction. EMSAs were run on a 1% Agarose gel containing 0.5 × Tris-Acetate-EDTA (TAE, Foothill Ranch, CA, USA) and a running buffer of 0.5 × TAE for 1 h at 80 V. Signals were visualized using a Thyphoon FLA 9500 instrument (GE Healthcare, Chicago, IL, USA) and analyzed with Quantity One software (Bio-Rad, Hercules, CA, USA). For determination of KD values, the intensity of the “free DNA” was measured using QuantityOne. Data were analyzed by the nonlinear last squares estimate of R fitting data with the Hill equation.
Lightshift poly didc
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LightShift Poly (dIdC) is a laboratory product offered by Thermo Fisher Scientific. It serves as a non-specific competitor DNA for use in electrophoretic mobility shift assays (EMSAs). This product provides a consistent source of non-specific competitor DNA to help identify and characterize DNA-protein interactions.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Qiaquick nucleotide removal kit, by Qiagen (2 mentions)
Typhoon fla 9500 laser scanner, by GE Healthcare (2 mentions)
Thyphoon fla 9500 instrument, by GE Healthcare (1 mentions)
Q5 high fidelity polymerase, by New England Biolabs (1 mentions)
Phosphor imaging plate, by Fujifilm (1 mentions)
γ 32p datp, by PerkinElmer (1 mentions)
Typhoon fla 7000, by Fujifilm (1 mentions)
Model 583 gel dryer, by Bio-Rad (1 mentions)
Oligo clean concentrator kit, by Zymo Research (1 mentions)
T4 polynucleotide kinase, by Zymo Research (1 mentions)
Streptavidin agarose bead, by Thermo Fisher Scientific (1 mentions)
Typhoon fla 9500, by GE Healthcare (1 mentions)
9 protocols using lightshift poly didc
1
Quantitative Analysis of rne-Cyanophage Binding
2
Radiolabeled DNA-Protein Binding Assay
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sAvL1-451 DNA were 5′-end-labeled with [γ−32P]dATP (PerkinElmer) using T4 polynucleotide kinase (NEB) and purified from excess of radioactive nucleotides using Oligo Clean & Concentrator kit (Zymo Research) following the manufacturer’s protocols. Binding reactions were set up in 10 µl total volume in a buffer with final concentrations 100 mM KCl, 10 mM Tris, pH7.4, 0.1 mM EDTA, 0.1 mM DTT, supplied with 500 ng LightShift™ Poly (dI-dC) (Thermo Scientific). Addition of 2.5 µl of AvMBD proteins provided 5% glycerol per reaction. Proteins were first pre-incubated with non-radioactive DNA for 15 min at RT. Then, 32P-labeled DNA was added to a final concentration of 0.05 nM, and reactions were incubated for additional 30 min at RT. After supplying with 6× EMSA gel-loading solution (Thermo Scientific), samples were loaded onto 6% DNA Retardation gels. Samples were run at 90 V in 0.5× TBE buffer (44.5 mM Tris–HCl, pH 8.3, 44.5 mM boric acid and 1 mM EDTA) at 4 °C for 90 min. Gels were dried using Model 583 Gel Dryer (BioRad), exposed with phosphorimaging plate (Fujifilm), scanned on Typhoon FLA 7000, and analyzed using Image Quant TL v8.1 software.
3
Nuclear Protein Extraction and EMSA Analysis
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Nuclear proteins from U87MG and NHA cells were extracted using a hypotonic lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCL) supplemented with DTT and protease inhibitors, followed by an extraction buffer (20 mM HEPES, ph 7.9, 1.5 mM MgCL2, 0.42 M NaCl, 0.2 mM EDTA, 25% v/v glycerol) supplemented with DTT and protease inhibitors. EMSA probes were designed to cover each SNP plus or minus 20 base pairs, for both major and minor alleles (Supplementary Table 4 ). Probe pairs were dissolved in TE buffer and annealed at a concentration of 10 μM each. Probes were labeled with ATP [γ-32P] (Perkin Elmer) using T4 polynucleotide kinase (NEB) and cleaned using the QiaQuick Nucleotide Removal Kit (Qiagen). Labeled probes were then incubated with protein extracts using LightShift Poly(dI-dC) (Thermo) and a binding buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.4) and electrophoresed on a 6% acrylamide gel overnight at 83 V. Gels were dried and films were exposed for 4–24 h. EMSAs were performed in two technical replicates.
4
Nuclear Protein Extraction and Electrophoretic Mobility Shift Assay
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Nuclear proteins from MCF10A and CAL51 cells were extracted using a hypotonic lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCL) supplemented with DTT and protease inhibitors, followed by an extraction buffer (20 mM HEPES, ph 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 25% v/v glycerol) supplemented with DTT and protease inhibitors. Electrophoretic mobility shift assays probes were designed to cover each SNP ±20 base pairs, for both major and minor alleles. Probe pairs were dissolved in water and annealed at a concentration of 10 μM each. Probes were labelled with ATP (γ-32 P; Perkin Elmer) using T4 polynucleotide kinase and cleaned using the QiaQuick Nucleotide Removal Kit (Qiagen). Labelled and unlabelled probes were then incubated with protein extracts using LightShift Poly(dI–dC) (Thermo) and a binding buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.4) and electrophoresed on a 6% acrylamide gel overnight at 83 V. Gels were dried and films were exposed for 4–24 h. Probe sequences are shown in Supplementary Table 21 .
5
DNA Pull-Down Assay Protocol
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DNA pull-down assays were carried out following a previous description34 (link). HeLa cells were lysed by sonication in HKMG buffer (10 mM HEPES, pH 7.9, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT and 0.5% NP-40) containing protease and phosphate inhibitors. Cellular debris was removed by centrifugation. Two milligrams of cell extract was precleared with 40 μl of streptavidin-agarose beads (Thermo Scientific, 20347) for 1 h at 4 °C and then incubated with 2 μg of biotinylated double-stranded oligonucleotides and 40 μg of LightShift Poly(dI-dC) (Thermo Scientific, 20148E) for 16 h at 4 °C. DNA-bound proteins were collected with 60 μL of streptavidin-Sepharose beads for 1 h at 4 °C, washed twice with HKMG buffer, separated by SDS-PAGE, and identified by western blot. The biotinylated double-stranded oligonucleotides were amplified using 5’ terminal biotinylated primers with the same sequences used for ChIP detection.
6
Quantification of DNA-Protein Interactions
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Cy3-labeled probes were amplified by PCR using the primers listed in Table S1 and purified with the Macherey-Nagel NucleoSpin Gel and PCR Clean-up Kit. Reaction conditions for binding protein to DNA were 12 mM HEPES, 4 mM Tris, 12 mM KCl, 1 mM EDTA, 1 mM EGTA, 12% glycerol, and 1 μg of LightShift Poly (dIdC) (Thermo Fisher Scientific). Samples were run on 3% agarose gels in 0.5x TBE (45 mM Tris, 45 mM boric acid, and 1 mM EDTA, pH 8.0) and visualized using Typhoon FLA 9500 (GE Healthcare) and Quantity One software (Bio-Rad).
The equilibrium dissociation constant (KD) between DNA probe and protein was calculated based on the intermediate lanes of the EMSAs. KD was calculated as , where the concentration of DNA-protein complex was calculated according to the relative signal intensity versus the control lane (without proteins). When available, two KD values were calculated for each DNA-protein pair, and the mean was used as the final KD value. Higher KD value indicates lower affinity between the DNA probe and the protein.
The equilibrium dissociation constant (KD) between DNA probe and protein was calculated based on the intermediate lanes of the EMSAs. KD was calculated as , where the concentration of DNA-protein complex was calculated according to the relative signal intensity versus the control lane (without proteins). When available, two KD values were calculated for each DNA-protein pair, and the mean was used as the final KD value. Higher KD value indicates lower affinity between the DNA probe and the protein.
7
Electrophoretic Mobility Shift Assay Protocol
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Cy3 end-labelled probes for EMSA were generated using primers #4–12 (Supplementary Table S1 ). Reaction conditions for binding protein to DNA were 12 mM HEPES, 15.7 mM Tris, 1 mM EDTA, 1 mM EGTA, 40.16% glycerol, 1 μg LightShift Poly (dIdC) (Thermo Fischer Scientific), 60 mM KCl, 64.2 mM NaCl, and 15.625 nM DNA probe. The concentration of proteins used for shift experiments were 595.4, 426.5, 297.3, 193.4, and 128.4 nM. Samples were run on 1.5% agarose gels in 0.5 × TAE.
8
CrhR-RNA Binding Assay
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Binding of different amounts of recombinant CrhR (0.1 to 5 pmol) to 0.2 pmol of Cy3 labelled RNA was performed in 20 mM HEPES-KOH (pH 8.3) buffer containing 3 mM MgCl 2 , 1 mM DTT, 500 μg/ml BSA. As a substrate competitor, 1 μg of LightShift Poly (dIdC) (Thermo Fischer Scientific) was included in each assay. The reaction was incubated at room temperature for 15 min prior to separation on 2% agarose-TAE gels.
The signals were visualized with a Laser Scanner Typhoon FLA 9500 (GE Healthcare) using a green light laser at a wavelength of 532 nm and a Cy3 filter (LPG, DGR1, BPG1.
The signals were visualized with a Laser Scanner Typhoon FLA 9500 (GE Healthcare) using a green light laser at a wavelength of 532 nm and a Cy3 filter (LPG, DGR1, BPG1.
9
CrhR-RNA Binding Assay
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Binding of different amounts of recombinant CrhR (0.1 to 5 pmol) to 0.2 pmol of Cy3 labelled RNA was performed in 20 mM HEPES-KOH (pH 8.3) buffer containing 3 mM MgCl 2 , 1 mM DTT, 500 μg/ml BSA. As a substrate competitor, 1 μg of LightShift Poly (dIdC) (Thermo Fischer Scientific) was included in each assay. The reaction was incubated at room temperature for 15 min prior to separation on 2% agarose-TAE gels.
The signals were visualized with a Laser Scanner Typhoon FLA 9500 (GE Healthcare) using a green light laser at a wavelength of 532 nm and a Cy3 filter (LPG, DGR1, BPG1.
The signals were visualized with a Laser Scanner Typhoon FLA 9500 (GE Healthcare) using a green light laser at a wavelength of 532 nm and a Cy3 filter (LPG, DGR1, BPG1.
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