Southern blot to detect intracellular capsid‐associated HBV DNA was performed as described (Ko et al,2018 ). Briefly, after cell lysis (50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1% NP‐40), samples were incubated for 15 min on ice and centrifuged at 15,322 g for 15 min at 4 °C. Supernatants were adjusted to 11 mM MgCl2, 200 µg/ml RNaseA, and 0.02 U/ml DNase I, incubated at 37°C for 3 h, and centrifuged at 15,322 g for 15 min at 4°C. HBV capsids were precipitated from supernatants with PEG8000 overnight on ice. After centrifugation (15,322 g, 15 min, 4°C), viral DNA was extracted from the pellet using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Viral DNA forms were separated on a 1.3% agarose gel, transferred to a nylon membrane (Hybond‐XL, Amersham Biosciences), hybridized with a digoxigenin‐labeled HBV probe, and developed with the DIG Luminescent Detection Kit (Roche). Northern blot was performed as described before (Ko et al,2018 ). Briefly, total RNA was extracted from cell culture using TRIzol Reagent (Ambion/Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions, separated through a 1% formaldehyde agarose gel, blotted onto a nylon membrane (Hybond‐XL, Amersham Biosciences), and hybridized as described above.
Hybond xl
Manufactured by Cytiva
Sourced in United States
Hybond-XL is a nitrocellulose membrane used for protein blotting, designed for high-performance applications. It provides an effective matrix for the transfer and immobilization of proteins from electrophoresis gels.
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Lab products found in correlation
Phosphoimaging screens, by GE Healthcare (2 mentions)
Luminata hrp substrate, by Merck Group (2 mentions)
Anti mouse hrp conjugated secondary antibody, by Merck Group (2 mentions)
Bio dot apparatus, by Bio-Rad (2 mentions)
Blood cell culture dna midi kit, by Qiagen (1 mentions)
Lightshift chemiluminescent emsa, by Thermo Fisher Scientific (1 mentions)
Super rx, by Fujifilm (1 mentions)
Cdp star, by Roche (1 mentions)
Decalabel dna labeling kit, by Thermo Fisher Scientific (1 mentions)
Decaprime 2 kit, by Thermo Fisher Scientific (1 mentions)
Purelink genomic dna extraction, by Thermo Fisher Scientific (1 mentions)
Semi dry trans blot apparatus, by Bio-Rad (1 mentions)
Fla 3000 series, by Fujifilm (1 mentions)
Dig luminescent detection kit, by Roche (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Amersham rediprime 2 dna labeling system, by GE Healthcare (1 mentions)
G 25 column, by GE Healthcare (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Hybond n, by Cytiva (1 mentions)
27 protocols using hybond xl
1
Intracellular HBV DNA Detection by Southern Blot
2
2D Gel Electrophoresis of DNA Replication Intermediates
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Genomic DNA was isolated using the QIAGEN Blood & Cell Culture DNA Midi Kit following the manufacturer's instructions. Then, the purified DNA was digested using XbaI and EcoRI, and separated by neutral/neutral 2D agarose gel electrophoresis as previously described (58 (link)). The DNA was transferred to a nylon membrane (Amersham Hybond XL) for Southern Blotting. The fragments of interest were detected with a P32 labelled DNA probe. Replication intermediates were quantified by calculating the percentage radioactivity signals in specific recombination intermediates relatively to the monomer spot as described (59 (link)).
3
Quantitative Analysis of NF-κB Activation
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NF-κB activation was analyzed by EMSA. Five μg of protein from the nuclear fraction was incubated with biotinylated double-stranded NF-κB oligonucleotide 5′-TTGTTACAAGGGACTTTCC GCTGGGGACTTTC GGGAGGCGTGG-3′; underlining indicates the NF-κB binding site, following supplier instructions (Cat. # 20148X, LightShift Chemiluminescent EMSA, Thermo Fisher Scientific). The DNA-protein complex was resolved on 6% nondenaturing polyacrylamide gel at 100 V in TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.3). DNA-protein complexes were electrotransferred onto a nylon membrane (Hybond-XL Amersham Pharmacia Biotech), and DNA was crosslinked to the membrane with a transilluminator (UVP model 2UV) at 302 nm for 15 min. After crosslinking, the membranes were blocked for 15 min and then incubated with streptavidin-HRP conjugate for 15 min and reactive bands were detected by chemiluminescence. Blot images were digitally acquired with an HP ScanJet 4670 scanner, and densitometry analysis of images was performed with the GelQuant.Net software. The results were expressed as integrated optical density (IOD).
4
Isolation and Analysis of Total RNA
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Total RNA was isolated by grinding 1.0 g of young leaves collected from 3–4 month-old plants in liquid N39 ,78 (link). For northern blot analysis, RNA (15.0 μg per lane) was fractionated on 1.6% formaldehyde agarose denaturing gels in HEPES buffer and blotted onto nylon membranes (Amersham Hybond-XL) in 10x SSC75 (link). Pre-hybridization, BvLzm probe labeling, hybridization, washing and detection of RNA gel blots were performed as described for Southern blot analysis.
5
Yeast Telomere Length Analysis
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Genomic DNA was isolated from yeast strains grown in YPAD for 3 days with or without the addition of 10 mM CaCl2. DNA was digested with XhoI, separated on a 1% agarose-Tris borate EDTA gel, transferred to a Hybond XL (Amersham Biosciences) membrane, and hybridized with a 32P-labeled DNA probe specific for the terminal Y’ telomere fragment. The probe was generated by random hexanucleotide-primed DNA synthesis using a short Y’-specific DNA template, which was generated by PCR from genomic yeast DNA using the primers Y up (5’-TGCCGTGCAACAAACACTAAATCAA -3’) and Y’ low (5’-CGCTCGAGAAAGTTGGAGTTTTTCA -3’). Two biological replicates of the whole experiment were conducted.
6
2D Gel Electrophoresis for DNA Analysis
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Neutral-neutral 2D gel electrophoresis was performed as previously described [40] (link). Approximately 3–10 µg of BND cellulose-enriched DNA was loaded for each 2D gel experiment. The first dimension gel (0.4% agarose) was run in 1X TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA) at 1.5 V/cm for 20 h at RT. The second dimension gel (1% agarose) was run in 1X TBE buffer (90 mM Tris, 90 mM Boric acid, 2 mM EDTA) containing 0.5 mg/ml ethidium bromide at 3 V/cm for 18 h at 4°C. DNA was transferred overnight to a charged nylon membrane (Hybond-XL, Amersham) in alkaline buffer by capillary blotting. Membranes were prehybridized at 37°C for 4 h in 1M NaCl, 1% SDS, 10% dextran sulfate, 5 mM Tris [pH 7.5], 100 µg/ml of denatured salmon sperm DNA and 25% formamide. rDNA 5′NTS probes were labeled by random priming and added directly to the prehybridization solution. After 18 h membranes washed 3 times in 2X SSC/1% SDS solution for 15 min each at 42°C, and once in 0.4X SSC/0.1% SDS solution for 15 min at 42°C. Blot were exposed to X-ray film with an intensifying screen at −70°C or analyzed with a phosphorimager.
7
RNA Extraction and Northern Blot
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RNA was extracted with Trizol reagent following a standard protocol. Three micrograms of total RNA were separated on a 1.2% agarose/formaldehyde gel for 1.5 h at 200 V. For northern blot analysis, rRNA were blotted onto a Hybond-XL (Amersham, UK) membrane by capillary transfer and probed for 20S (5′-GGTTTTAATTGTCCTATAACAAAAGC) using radioactively labeled probes (synthesized by Microsynth). rRNA was detected using phosphoimaging screens (GE Healthcare).
8
Molecular Detection of Plant Viral Infections
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Total genomic DNA was extracted from inoculated and control plants by the CTAB method (Doyle and Doyle, 1990 ). TA was detected by PCR using CP or AV2 primers and CLCuMuB using primers beta01/beta02 (Table 1 ). For Southern blotting, 10 μg of DNA extracted from plants was electrophoresed in 1.5% agarose gels and then transferred to nylon membranes (Hybond XL, Amersham) by capillary transfer (Sambrook et al., 1989 ). TA was detected using PCR-amplified, digoxigenin (DIG)-labeled (Roche, Germany) probes to the IR (primers ToLNC4pvx/35R and ToLNV2pvx/35R) and/or TrAP gene (primers ToLNC2pvx/35F and ToLNC2pvx/35R), whereas CLCuMuB (Cβ) was detected using a βC1 gene probe (primers BetaC1F/BetaC1R). ToLCNDV DNA B (TB) was detected on Southern blots using a PCR amplified radioactively labeled (MBI Fermentas, DecaLabel ™ DNA Labeling Kit) MP gene probe as described previously (Dalakouras et al., 2009 (link)). Hybridization was conducted at 50°C for 16 h and signals were detected on X-ray film (Super RX, Fuji film) after treating with CDP-Star (Roche, Germany), while a phosphoimager (PharosFX™ Systems Life Science Research Bio-Rad) was used to detect radioactive signals on blots.
9
Northern Blot Analysis of RDN18 and FLO11
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Total RNA from the cells of colonies grown on GMA plates was isolated by the hot phenol method as previously described [14 (link)]. For northern blots, 12 μg of total RNA was separated on a 1.5% agarose gel, transferred to a positively charged nitrocellulose membrane (Hybond-XL, Amersham Bioscience) and exposed to a labeled probe. The radioactive signal was visualized on Fuji X-ray film. The DNA probe for the RDN18 gene was a complete ORF of the gene prepared by PCR reaction. For the FLO11 probe, a PCR fragment corresponding to the last 1382 bp of the FLO11 gene was used. The [α-32P] dCTP-labeled probes were obtained by random priming using the DecaPrime II Kit (Ambion).
10
Dot Blot Assay for DNA Modifications
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Ten nanograms denatured oligos (CG-containing oligos: 5′-GCATCGTACGGAATCGCTTCTAGCCGGACATTAGCGATX GATCGATCAGGCTCGTAGGTACTCGACGGCAATCGTTAG-3′ or (CA)9-containing oligos: 5′-CTAACGATTGCCGTCGCACACACAX ACACACACAGATCGCTAATGTCCGC-3′; X = C, 5mC, 5hmC, 5fC, or 5caC) were spotted onto a positively charged membrane (Amersham Hybond-XL). The membrane was then baked at 80°C and blocked for 1 h with 5% nonfat milk in TBST buffer (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20). Membranes were then incubated overnight with a 1:500 dilution of 5mC, 5hmC, 5fC, or 5caC antibodies. After three rounds of washing with the blocking solution, membranes were incubated with a 1:20,000 dilution of HRP-conjugated anti-mouse (for 5mC) or anti-rabbit (for 5hmC, 5fC, and 5caC) IgG secondary antibody. The membranes were then washed with TBST and treated with ECL.
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