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Poly di dc

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Poly(dI-dC) is a synthetic DNA polymer composed of alternating deoxycytidine and deoxyinosine nucleotides. It is commonly used as a non-specific competitor DNA in various molecular biology applications, such as gel shift assays and chromatin immunoprecipitation experiments.

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Lab products found in correlation

X ray film, by GE Healthcare (6 mentions) Nuclear extract kit, by Active Motif (3 mentions) Anti flag m5, by Merck Group (3 mentions) Sephadex g 25m column, by GE Healthcare (2 mentions) T4 kinase, by Thermo Fisher Scientific (2 mentions) 32p atp, by PerkinElmer (2 mentions) Tnt quick coupled transcription translation system, by Promega (2 mentions) Oligonucleotide, by GE Healthcare (2 mentions) Oligonucleotides for nf κb, by Promega (2 mentions) Ecl plus system, by GE Healthcare (1 mentions) Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions) T4 polynucleotide kinase, by Takara Bio (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Goat anti rabbit igg h l hrp conjugate, by Bio-Rad (1 mentions) Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions) Amv rt, by Promega (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) 17β estradiol, by Merck Group (1 mentions) X ray film, by Kodak (1 mentions)

17 protocols using poly di dc

1

Estrogen Receptor Binding Assay

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Trizol reagent (Invitrogen) was used for total RNA preparation. AMV RT was from Promega. Polymerases, restriction and modifying enzymes were from New England Biolabs, Qiagen and Invitrogen. Plasmids were prepared using Qiagen kits, and clones were sequenced with BigDye Terminator v3.1 (Applied Biosystems). 17-β-estradiol was from Sigma. Estradiol [2,4,6,7,16,17-3H(N)] (152 Q3 Ci/mmol), γ-32P labeled ATP and poly dI-dC ○ poly dI-dC were purchased from GE Healthcare.
Kundu P., Li M., Lu R., Stefani E, & Toro L. (2014). Regulation of transcriptional activation function of rat estrogen receptor α (ERα) by novel C-terminal splice inserts*. Molecular and cellular endocrinology, 0, 202-212.
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2

Electrophoretic Mobility Shift Assay Protocol

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EMSA was performed as previously described (35 (link)). Briefly, nuclear protein extracts were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA), following the manufacturer’s protocol. Probe labeling was accomplished by treatment with T4 kinase (Life Technologies, Burlington, ON, Canada) in the presence of [32P]ATP (Perkin Elmer, Waltham, MA). Labeled oligonucleotides were purified on a Sephadex G-25M column (GE Healthcare, Pittsburgh, PA). Nuclear protein (10 μg) was added to a 10-μl volume of binding buffer supplemented with 1 μg poly(dI:dC) (GE Healthcare) for 15 min at room temperature. Labeled double-stranded oligonucleotide was added to each reaction mixture, incubated at room temperature for 30 min, and separated by electrophoresis on a 6% polyacrylamide gel in 0.5× Tris-boric acid-EDTA buffer. Gels were vacuum-dried and subjected to autoradiography. The following synthesized double-stranded oligonucleotides were used: NF-κB consensus sequence on the IL-6 promoter (5′-AGTTGAGGGGACTTTCCCAGGC-3′) (Promega, Madison, WI) and NFAT-binding consensus sequence on the mouse IL-13 promoter (5′-AAGGTGTTTCCCCAAGCCTTTCCC-3′) (Sigma-Aldrich).
Pang Z., Raudonis R., McCormick C, & Cheng Z. (2019). Early Growth Response 1 Deficiency Protects the Host against Pseudomonas aeruginosa Lung Infection. Infection and Immunity, 88(1), e00678-19.
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3

Molecular Reagents for Cell Biology

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ATRA, DMSO, and mouse anti-β-actin monoclonal antibody were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Buprenorphine hydrochloride was purchased from Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). Pentobarbital sodium salt was purchased from Tokyo Kasei Co., Ltd. (Tokyo, Japan). Anti-Npt2b antibody was purchased from Alpha Diagnostics (San Antonio, TX, U.S.A.). Goat anti-rabbit IgG(H + L)-HRP conjugate was purchased from Bio-Rad (Hercules, CA, U.S.A.). ECL Plus system and poly(dI-dC) were purchased from GE Healthcare (Buckinghamshire, U.K.). QuikChange® site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA, U.S.A.). 4-[E-2-(5, 6, 7, 8-Tetrahydro-5, 5, 8, 8-tetra-methyl-2-naphtalenyl)-1-propenyl] benzoic acid (TTNPB) was purchased from Biomol Research Laboratories (Boston, MA, U.S.A.). Double-strand Stealth RNAi oligos for C/EBPβ and negative control were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.). [γ−32P] ATP was purchased from ICN (Costa Mesa, CA, U.S. A.). TNT® Quick Coupled Transcription/Translation System was purchased from Promega Corporation (Madison, WI, U.S.A.). T4 polynucleotide kinase was purchased from Takara (Shiga, Japan). C/EBP consensus oligonucleotide (cebp; catalog number sc-2525) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).
Masuda M., Yamamoto H., Takei Y., Nakahashi O., Adachi Y., Ohnishi K., Ohminami H., Yamanaka-Okumura H., Sakaue H., Miyazaki M., Takeda E, & Taketani Y. (2020). All-trans retinoic acid reduces the transcriptional regulation of intestinal sodium-dependent phosphate co-transporter gene (Npt2b). The Biochemical journal, 477(4), 817-831.
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4

Biotinylated Probe Preparation and EMSA

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The PCR primers and template DNA used for preparing biotinylated probes are shown in Supplementary Tables S1, S2-2. Site-directed mutagenesis of the probes was performed using an oligonucleotide-based PCR method as described previously (Ogura and Tanaka, 1996 (link)). For EMSA, appropriate amounts of SinR and (or) RNAP were incubated for 15 min at 28°C with a probe (20 fmol) in 16 μl of a reaction mixture (15 mM Tris-Cl, 4 mM MOPS-KOH, 15 mM KCl, 50 mM NaCl, 0.8 mM MgCl2, 0.6 mM DTT, and 12.5% glycerol, pH 7.8) containing 1 μg of poly(dI-dC) (GE Healthcare). After the addition of 2 μl of loading buffer [40% glycerol, 1× TBE (89 mM Tris-borate, and 2 mM EDTA, pH 8), 2 μg/ml bromophenol blue], the samples were applied onto a polyacrylamide gel, and electrophoresis was performed in 0.1× TBE buffer at 4°C. The method used for the detection of biotin-labeled DNA was described previously (Ogura and Tanaka, 1996 (link)).
Most EMSAs were performed with the gradient of the SinR concentration. Not a few critical EMSAs were duplicated.
Fujita Y., Ogura M., Nii S, & Hirooka K. (2017). Dual Regulation of Bacillus subtilis kinB Gene Encoding a Sporulation Trigger by SinR through Transcription Repression and Positive Stringent Transcription Control. Frontiers in Microbiology, 8, 2502.
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5

NF-κB DNA Binding Assay

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The binding mixture contained radiolabeled 32P-NF-κB oligonucleotide probe (Promega), 10 µg of cell extracts, 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 500 ng of poly (dI-dC) (GE Healthcare), and 10 µg of bovine serum albumin to a final volume of 15 µl. The reaction mixture was incubated at 25°C for 30 min, separated by 5% (w/v) polyacrylamide gel, and visualized by autoradiography.
Ikeda Y., Morioka S., Matsumoto K, & Ninomiya-Tsuji J. (2014). TAK1 Binding Protein 2 Is Essential for Liver Protection from Stressors. PLoS ONE, 9(2), e88037.
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6

NF-κB Binding Assay for IL-6 Promoter

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Nuclear protein extracts were prepared using a nuclear extract kit (Active Motif). EMSA was performed using a 32P-labled oligonucleotide probe specific for the NF-κB consensus sequence on the IL-6 promoter [57 (link)]. Briefly, 10 μg of nuclear protein was added to 10 μl of binding buffer supplemented with 1 μg poly-(dI-dC) (GE Healthcare) and incubated at room temperature for 15 minutes before mixing with the probe. The reaction mixture was incubated at room temperature for 30 minutes and subjected to electrophoresis on 6% polyacrylamide gels. Gels were vacuum-dried and subjected to autoradiography.
Yan C., Lei Y., Lin T.J., Hoskin D.W., Ma A, & Wang J. (2017). IL-17RC is critically required to maintain baseline A20 production to repress JNK isoform-dependent tumor-specific proliferation. Oncotarget, 8(26), 43153-43168.
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7

EMSA with RBPJ Binding Assay

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Reticulocyte lysates from in vitro translations were used for electromobility shift assays (EMSAs) in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol. For the binding reaction, 10 ng (0.02 U) poly(dI-dC) (GE healthcare) and approximately 0.5 ng of 32P-labeled oligonucleotides were added. The sequence of the double-stranded oligonucleotide with 2 RBPJ binding sites (underlined): 5´-CCT GGA ACT ATT TTC CCA CGG TGC CCT TCC GCC CAT TTT CCC ACG AGT CG-3 and reverse strand: 5´-CTC GCG ACT CGT GGG AAA ATG GGC GGA AGG GCA CCG TGG GAA AAT AGT TC-3´. Super shifting of complexes was achieved by adding 1 μg of anti-Flag (M5, Merck) antibody. The reaction products were separated using 5% polyacrylamide gels with 1x Tris-glycine-EDTA at room temperature. Gels were dried and exposed to X-ray films (GE Healthcare).
Gagliani E.K., Gutzwiller L.M., Kuang Y., Odaka Y., Hoffmeister P., Hauff S., Turkiewicz A., Harding-Theobald E., Dolph P.J., Borggrefe T., Oswald F., Gebelein B, & Kovall R.A. (2022). A Drosophila Su(H) model of Adams-Oliver Syndrome reveals cofactor titration as a mechanism underlying developmental defects. PLoS Genetics, 18(8), e1010335.
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8

Electrophoretic Mobility Shift Assay for AE9a Binding

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Transfected HEK-293 cells were lysed using a freeze–thaw method in a lysis buffer containing 0.2 mM EDTA, 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl and 25% glycerol. Six micrograms of protein lysate was incubated with 1.5 μg poly(dI-dC) (GE Healthcare, Chalfont St Giles, UK) and approximately 0.5 ng of [32P]-dCTP-labeled oligonucleotides in a binding buffer consisting of 0.5 mM DTT, 5 mM EDTA, 5 mM MgCl2, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5) and 25% glycerol. The sequence of the double-stranded oligonucleotide Runt2X (5′-CTAGAGAGTGGTGTGGTAGTGCGGTCGGGGT-3′) corresponds to the AE9a-binding sites published in Okumura et al.36 (link) Super shifting of complexes was achieved by adding 1 μg of anti-Flag antibody (M5, Sigma-Aldrich, #F4042). The reaction products were resolved at room temperature in a 7.5% PAGE with 0.5 × Tris-Borate-EDTA. Gels were dried and exposed to X-ray films (GE Healthcare).
Thiel V.N., Giaimo B.D., Schwarz P., Soller K., Vas V., Bartkuhn M., Blätte T.J., Döhner K., Bullinger L., Borggrefe T., Geiger H, & Oswald F. (2017). Heterodimerization of AML1/ETO with CBFβ is required for leukemogenesis but not for myeloproliferation. Leukemia, 31(11), 2491-2502.
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9

EMSA Analysis of RBPJ-Binding to EBV TP-1 Promoter

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Reticulocyte lysates from in vitro translations were used for electromobility shift assays (EMSAs) in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol. For binding reaction, 2 μg poly(dI-dC) (GE healthcare) and approximately 0.5 ng of 32P-labeled oligonucleotides were added. The sequence of the double-stranded oligonucleotide FO-233 (Key Resources Table) corresponds to the two RBPJ-binding sites within the EBV TP-1 promoter. Super shifting of complexes was achieved by adding 1 μg of anti-Flag (M5, Sigma) antibody. The reaction products were separated using 5% polyacrylamide gels with 1x Tris-glycine-EDTA at room temperature. Gels were dried and exposed to X-ray films (GE Healthcare).
Yuan Z., VanderWielen B.D., Giaimo B.D., Pan L., Collins C.E., Turkiewicz A., Hein K., Oswald F., Borggrefe T, & Kovall R.A. (2019). Structural and Functional Studies of the RBPJ-SHARP Complex Reveal a Conserved Corepressor Binding Site. Cell reports, 26(4), 845-854.e6.
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10

Electrophoretic Mobility Shift Assay for NF-κB and SREBP

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Oligonucleotides for NF-κB (5′-AGTTGAGGGGACTTTCCCAGG-3′) was purchased from Promega. Oligonucleotides for sterol regulatory element (SRE), wild type (5’-TTTGAAAATCACCCCACTGCAAACTCC-3’) and mutant (5’-TTTGAAAGTCAAACCGTTGCAAACTCC-3’) sterol regulatory element (SRE) were synthesized.68 (link), 69 (link) The binding reaction contained 32P radiolabeled oligonucleotide probe, 10 μg of cell extracts, 4% glycerol, 1mM MgCl2, 0.5mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 500 ng of poly (dI-dC) (GE Healthcare), and 10 μg of bovine serum albumin to a final concentration of 15 μl. The reaction mixture were incubated at 25oC for 30 min, separated by 5% (w/v) polyacrylamide gel, and visualized by autoradiography. The NF-κB band was confirmed by super-shift using anti-p65 NF-κB. Since antibodies against SREBPs are not suitable for super-shift of mouse SREBP, the SREBP band was confirmed by a completion with wild type and mutant SRE.
Morioka S., Sai K., Omori E., Ikeda Y., Matsumoto K, & Ninomiya-Tsuji J. (2016). TAK1 regulates hepatic lipid homeostasis through SREBP. Oncogene, 35(29), 3829-3838.
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