Dna retardation gel

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The 6% DNA retardation gel is a laboratory equipment used for the separation and analysis of DNA molecules. It is a polyacrylamide-based gel that provides a consistent and reliable medium for the electrophoretic separation of DNA fragments. The gel's 6% concentration is suitable for the effective resolution of a wide range of DNA fragment sizes, making it a versatile tool for various DNA-based applications.

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70 protocols using dna retardation gel

Characterizing Transcriptional Regulation by EMSA

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Electrophoretic mobility shift assays were performed as previously described (Kim et al., 2010 (link)). The promoter region of the SO_3165–3166 operon (296 nt) (PSO_3165–3166) was amplified by PCR from genomic DNA using the Pfu DNA polymerase from genomic DNA with primers PSO_3165-f and PSO_3165-r. The PCR products were gel purified with a QIAquick Gel Extraction Kit (Qiagen) and labeled with biotin using the Biotin 3′-end DNA Labeling Kit (Pierce). For the binding reactions, biotin-PSO_3165–3166 (0.05 pmol) DNA was incubated with purified SO_3165-CHis protein for 1 h at room temperature. The binding reaction conditions were performed with the non-specific competitor DNA (poly dI-dC) and NP-40 in buffer containing 10 mM HEPES (pH 7.3), 20 mM KCl, 1 mM MgCl₂, and 5% glycerol at 25°C for 1 h. The samples were run on a 6% DNA retardation gel (Invitrogen) at 100 V in 0.5 × TBE (10 mM Tris borate at pH 8.3 and 2 mM EDTA) for 90 min. Then, the DNA was transferred to a nylon membrane at 390 mA for 45 min, followed by UV cross-linking at 302 nm. Chemiluminescence was performed with the LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Rockford, IL) according to the manufacturer's protocol.

Yao J., Guo Y., Zeng Z., Liu X., Shi F, & Wang X. (2015). Identification and characterization of a HEPN-MNT family type II toxin–antitoxin in Shewanella oneidensis. Microbial Biotechnology, 8(6), 961-973.

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Hemimethylated DNA Binding Assay

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A 26-bp hemimethylated DNA duplex (upper strand: 5′-ACACCAAGCCTGmCGGAGGCTCACGGA-3′, mC = 5-methyl-2′-deoxycytidine; lower strand: 5′-TCCGTGAGCCTCCGCAGGCTTGGTGT-3′) was used to characterize DNA binding of the wild-type hDNMT1_351–1600 and its R582E mutant. Binding reactions contained 0.1 μM dsDNA and various concentrations of protein, dissolved in 10 μl of binding buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1mM DTT). Binding reaction mixtures were electrophoresed in 6% DNA retardation gel (Invitrogen) in 0.25X TBE (89 mM Tris-borate, pH 8.4) buffer at 4 °C under 100 V for 50 min. DNA was visualized by SYBR green staining and quantitated with imageJ software (44 (link)).

Zhang Z.M., Liu S., Lin K., Luo Y., Perry J.J., Wang Y, & Song J. (2015). Crystal Structure of Human DNA Methyltransferase 1. Journal of molecular biology, 427(15), 2520-2531.

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Mn2+-Dependent DNA/RNA Binding Assay

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6 nM ³²P-labeled DNA/RNA oligo was incubated with various amount of TOP3B/TOP3Bcore, in 10 mM Tris-HCl (pH 8.0), 40 mM KCl, 0.01% Tween 20, 6% Glycerol, 0.01% BSA, 2 mM DTT, and requested concentration of divalent cations/EDTA, at 30 °C for 30 min. Buffer for RNA assay contains 1X RNasin® Plus Ribonuclease Inhibitor (Promega). Reaction sample was placed on ice for 5 min before loading on a pre-run 6% DNA retardation gel (Invitrogen) at 4 °C. Electrophoresis was conducted at 200 V for 45 min in the gel running buffer containing 0.5X TBE with MnCl₂ addition (Mn²⁺ concentration was adjusted to 0.1 mM).

Yang X., Saha S., Yang W., Neuman K.C, & Pommier Y. (2022). Structural and biochemical basis for DNA and RNA catalysis by human Topoisomerase 3β. Nature Communications, 13, 4656.

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Quantitative Protein-DNA Binding Assay

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Proteins were purified using Ni‐NTA and GST resin and quantified using Coomassie blue staining with BSA standards. DNA probes were generated using polynucleotide kinase to ³²P‐end label oligos, followed by annealing to their unlabeled complementary strands (Table S6). The fraction of active protein was similar for Rap1 and Rap1^SHY preparations (~85%), and was quantified by incubating 10 nM protein as measured by Coomassie blue staining with increasing amounts of TEF2 probe (0–100 nM), taking the fraction of protein–DNA complex formation at saturated DNA concentrations as a measure of active protein. For EMSAs, 0.5 nM ³²P‐labeled duplexes were incubated with increasing concentrations of active protein in binding buffer (20 mM Hepes‐KOH, pH 8, 100 mM KCl, 10 µg/ml BSA, 1 mM EDTA, 2 mM MgCl₂, 5% glycerol) for 30 min at room temperature. Reactions were loaded on 6% DNA retardation gel (Invitrogen) and electrophoresis was conducted at 100V at 4°C. Radioactive signals were visualized using Typhoon FLA 7000.

Song S., Perez J.V., Svitko W., Ricketts M.D., Dean E., Schultz D., Marmorstein R, & Johnson F.B. (2019). Rap1‐mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence. Aging Cell, 19(1), e13061.

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Gel Shift Assay for Protein-DNA Complexes

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5× TBE Hi-Density buffer (15% Ficoll (w/v), 5% glycerol (v/v), 1× TBE Buffer (Invitrogen, 15581044)) was added in a 1:4 ratio to assembled protein–DNA complexes and samples separated on a 6% DNA retardation gel (Invitrogen) at 100 V in 0.5× TBE Buffer for 45 min. The gel was incubated with 3 μl SYBR Green I (Sigma-Aldrich) diluted in 30 ml of 1× TBE Buffer and rocked for 20 min at room temperature. Excess SYBR Green I dye was washed off by rinsing the gel three times with MilliQ water. DNA was visualized on a LAS4000 Image Quant (GE Healthcare).

Reim A., Ackermann R., Font-Mateu J., Kammel R., Beato M., Nolte S., Mann M., Russmann C, & Wierer M. (2020). Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry. Nature Communications, 11, 3019.

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Amplification and Gel Shift Analysis of attDOT

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A 325-bp fragment of attDOT was amplified from pCMK937 containing attDOT (9 (link)) using oligonucleotides listed in Table 1. The reaction mixture contained attDOT DNA (100ng/μL), IntDOT crude extracts with 7ng of protein before dilution, 7nM purified BHFa, gel shift buffer [50mM Tris-HCl (pH 8), 1mM EDTA, 50mM NaCl, 10% glycerol, and 2μg/mL heparin sulfate] and dilutions of crude extracts of IntDOT and mutants with 7ng of protein before dilution. The samples were incubated at room temperature for 20 minutes before being loaded on an Invitrogen 6% DNA retardation gel and run at 100V and 20mA for 1 hour. The gels were incubated with Sybr Green for 40 minutes at room temperature before being analyzed by a Fuji phosphoimager.

Kolakowski A.J, & Gardner J.F. (2016). The N-terminus of IntDOT Forms Hydrophobic Interactions During Holliday Junction Resolution. Plasmid, 87-88, 10-16.

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RNA-Protein Binding Assay

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Purified LRPPRC, LRPPRC–SLIRP or SLIRP (0, 0.02, 0.04, 0.08, 0.16, 0.36, 0.64, 1.28, 2.56 μM) was incubated at room temperature for 15 min with 40 ng fluorescein labelled RNAs in 10 mM HEPES (pH 8.0), 1 mM EDTA, 60 mM NaCl, 3 mM MgCl₂, 1 mM DTT, 0.1 mg/ml fatty acid-free BSA and 5% glycerol. Reactions were separated by electrophoresis in a 6% DNA retardation gel (Invitrogen) in 0.5× TBE and fluorescence was detected using a Typhoon FLA 9500 biomolecular imager (GE).

Spåhr H., Rozanska A., Li X., Atanassov I., Lightowlers R.N., Chrzanowska-Lightowlers Z.M., Rackham O, & Larsson N.G. (2016). SLIRP stabilizes LRPPRC via an RRM–PPR protein interface. Nucleic Acids Research, 44(14), 6868-6882.

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EMSA for TATA Box Protein Binding

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EMSA was performed using annealed 18 bp oligonucleotides containing a TATA box at central position as probe. Freshly annealed probe was diluted at 2 nM in buffer A (HEPES 20 mM pH 7.9, MgCl₂ 8 mM, DTT 1 mM). Fresh PURExpress reaction mix (2 μl) was diluted in 8 μl of buffer B (Tris HCl 20 mM pH 7.9, KCl 100 mM, DTT 1 mM, BSA 50 μg/ml, glycerol 20%). Ten microliters of Buffer A-probe was mixed with 10 μl of TBP containing buffer B and incubated for 30 min at 30°C. Reaction mix was subsequently resolved by native electrophoresis on 6% DNA Retardation Gel (Invitrogen) with 0.5X TBE running buffer at 100V for 45 min. Gel was then imaged on a Typhoon 9400 (Amersham Biosciences) using appropriate Alexa 488 wavelength.

Hardivillé S., Banerjee P.S., Alpergin E.S., Smith D.M., Han G., Ma J., Talbot CC J.r., Hu P., Wolfgang M.J, & Hart G.W. (2019). TATA-Box Binding Protein O-GlcNAcylation at T114 regulates formation of the B-TFIID complex and is critical for metabolic gene regulation. Molecular cell, 77(5), 1143-1152.e7.

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Electrophoretic Mobility Shift Assay Protocol

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EMSA was performed according to LightShift Chemiluminescent EMSA Kit Protocols, (Thermo Scientific, United States). In brief, oligonucleotides b-WT (124 bp) from Borrelia burgdorferi strain B31 (Riley et al., 2009 (link)) and cc-WT (200 bp of ybab_C_c gene) from C. crescentus (this work) were labeled at 3′ end with Biotin-11-UTP according to the manufacturer’s protocol (Biotin 3′ end DNA Labeling Kit, Thermo Scientific, United States). Labeled target probes were incubated with increasing concentrations of purified YbaB_C_c in a reaction containing binding buffer (LightShift Chemiluminescent EMSA Kit, Thermo Scientific) and 50% glycerol at 25°C for 30 min. The reactions were electrophoresed in 6% DNA retardation gel (Invitrogen, United States) in 0.5x TBE. Separated DNA products were then electroblotted onto a positively charged Nylon membrane (Thermo Fisher Scientific, United States) using a semi-dry transfer apparatus (Bio-Rad, United States). Cross-linking by UV was done at 120 mJ/cm² for 1 min immediately after electroblotting. Detection of DNA and DNA-protein complexes was carried using Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific, United States).

Pal P., Modi M., Ravichandran S., Yennamalli R.M, & Priyadarshini R. (2021). DNA-Binding Properties of YbaB, a Putative Nucleoid-Associated Protein From Caulobacter crescentus. Frontiers in Microbiology, 12, 733344.

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Electrophoretic Mobility Shift Assay Protocol

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EMSAs were conducted as described (Lee and Gralla, 2001; Zhao et al., 2005). Briefly, DNA fragments were amplified using the primer pairs shown in Table S1. PCR amplicons were gel purified with a QIAquick Gel Extraction Kit (Qiagen), and the purified products were labelled with the Pierce™ biotin 3′ end DNA labelling kit (Thermo Fisher Scientific, Rockford, IL). The binding reaction was performed with the non‐specific competitor DNA (poly dI‐dC) and NP‐40 in buffer containing 10 mM HEPES (pH 7.3), 20 mM KCl, 1 mM MgCl₂ and 5% glycerol at 25°C for 2 h. The final mixtures were run on a 6% DNA retardation gel (Invitrogen), transferred to a nylon membrane and UV cross‐linked. Chemiluminescence was performed with the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

Guo Y., Li Y., Zhan W., Wood T.K, & Wang X. (2019). Resistance to oxidative stress by inner membrane protein ElaB is regulated by OxyR and RpoS. Microbial Biotechnology, 12(2), 392-404.

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