The detailed protocol has been reported previously [59 (link),60 (link)]. Briefly, after electrophoresis in 5% (w/v) polyacrylamide/8 M urea gel for 1 h at 200 V, RNAs were then transferred to Hybond-XL nylon membranes (Amersham Biosciences, Little Chalfont, United Kingdom) by a Bio-Rad semi-dry transfer cassette and were immobilized by a UV-crosslinker (UVP, Upland, CA). The RNAs were then detected by DIG-labeled UTP probes. PSTVd RNAs were prepared as described before [23 (link)]. SmaI-linearized pInt95-94(-) and pInt95-94(+) were used as templates for generating probes, using the MAXIscript kit (Thermo Fisher Scientific). The DIG-labeled probes were used for detecting PSTVd RNAs. RNA gel blot signals were obtained using ChemiDoc (Bio-Rad Laboratories) and quantified using ImageJ (https://imagej.nih.gov/ij/ ). The quantified signals with biological replicates were subject to graphing and statistical analysis using the built-in functions of Prism (GraphPad Software, LLC). The raw quantification data used for graphing were listed in S7 Dataset .
Hybond xl nylon membrane
Manufactured by Cytiva
Sourced in United Kingdom
Hybond-XL is a nylon membrane used in blotting techniques. It provides high-binding capacity for the transfer and immobilization of nucleic acids and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (12 mentions)
Chemidoc, by Bio-Rad (3 mentions)
Uv crosslinker, by Analytik Jena (2 mentions)
Semi dry transfer cassette, by Bio-Rad (2 mentions)
Prime it 2 random primer labeling kit, by Agilent Technologies (2 mentions)
Whatman, by Cytiva (2 mentions)
Maxiscript kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti mouse serum, by Bio-Rad (1 mentions)
Myecl imager, by Thermo Fisher Scientific (1 mentions)
Maxiscript t7 kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Mini protean tetra cell, by Bio-Rad (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
Dna blood tissue kit, by Qiagen (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Molecular imager fx, by Bio-Rad (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Phosphor imaging screen, by Bio-Rad (1 mentions)
Synthetic peptide, by Eurogentec (1 mentions)
12 protocols using hybond xl nylon membrane
1
RNA Gel Blot Analysis of PSTVd
2
Yeast Genomic DNA Quantification
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Total genomic DNA was extracted from Saccharomyces cerevisiae (strain BY4741) and quantified on gel. Two-fold serial dilutions of this genomic DNA were made in 0.4 M NaOH, incubated at room temperature for 10 min, then spotted on Hybond-XL nylon membranes (Amersham RPN 203S), before proceeding to hybridization.
3
Immunoblot and Northern Blot Analyses
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After electrophoresis, RNAs were then transferred to Hybond-XL nylon membranes (Amersham Biosciences, Little Chalfont, UK) via a semi-dry transfer cassette (Bio-Rad Laboratories) and were immobilized by a UV-crosslinker (UVP, Upland, CA, USA). RNAs were then detected by DIG-labeled UTP probes. AP-conjugated anti-DIG monoclonal antibody (catalog #11333089001; MilliporeSigma) was used in combination with Immun-Star substrates (Bio-Rad Laboratories). Signals were captured by ChemiDoc (Bio-Rad Laboratories).
After SDS–PAGE electrophoresis, we followed a previously described protocol for immunoblotting (Jiang et al., 2019 ). IMPas were detected by a monoclonal mouse anti-Myc antibody (catalog #M5546; MilliporeSigma; 1:3,000 dilution). VIRP1 was detected by a monoclonal mouse anti-FLAG antibody (catalog #F1804-200UG; MilliporeSigma; 1:1,000 dilution). HRP-conjugated anti-mouse serum (catalog #1706516; Bio-Rad Laboratories) was diluted at 1:2,000. SuperSignal West Dura (Thermo Fisher Scientific) was used as the substrate. Signals were captured by ChemiDoc (Bio-Rad Laboratories).
After SDS–PAGE electrophoresis, we followed a previously described protocol for immunoblotting (Jiang et al., 2019 ). IMPas were detected by a monoclonal mouse anti-Myc antibody (catalog #M5546; MilliporeSigma; 1:3,000 dilution). VIRP1 was detected by a monoclonal mouse anti-FLAG antibody (catalog #F1804-200UG; MilliporeSigma; 1:1,000 dilution). HRP-conjugated anti-mouse serum (catalog #1706516; Bio-Rad Laboratories) was diluted at 1:2,000. SuperSignal West Dura (Thermo Fisher Scientific) was used as the substrate. Signals were captured by ChemiDoc (Bio-Rad Laboratories).
4
RNA Blot Analysis of Plant Samples
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Leaf samples were collected from the top two fully expanded leaves at 28 days post-inoculation (dpi). Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). RNA blot was performed as previously described [16 (link)]. Roughly 10 mg of RNA samples were loaded onto a 5% polyacrylamide gel containing 8 M urea followed by electrophoresis using Bio-Rad Mini-PROTEAN Tetra Cell. These samples were then transferred onto a Hybond-XL nylon membrane (Amersham Biosciences, Piscataway, NJ, USA) using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad, Shanghai, China) and immobilized through UV-cross-linking. Hybridization was conducted at 65 °C using DIG-11-UTP-labeled riboprobes. These riboprobes were prepared through in vitro transcription using the T7 Maxiscript kit (ThermoFisher Scientific), with the SpeI-linearized pInter(–) plasmid serving as the template [19 (link)]. Following an overnight hybridization, the membranes were subjected to two rounds of washing at 65 °C, first in a solution of 2× SSC (where 1× SSC contains 0.15 M NaCl and 0.015 M sodium citrate) with 0.1% sodium dodecyl sulfate (SDS) and then in 0.2× SSC-0.1% SDS. Signal detection was accomplished using the MYECL™ Imager (Thermo Fisher Scientific, Shanghai, China).
5
Northern Blot Analysis of PSTVd RNA
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Total RNA isolated from floral organs and systemic leaves (5 μg) was separated in a 1.4% formaldehyde agarose gel. The gel was transferred to a Hybond-XL nylon membrane (Amersham Biosciences) overnight by capillary transfer, using 2 × saline sodium citrate buffer (SSC), and the RNA preparations were immobilized by UV cross-linking. The membrane was stained with methylene blue and then prehybridized with sodium dodecyl sulphate (SDS) hybridization buffer (7% SDS, 50% deionized formamide, 5 × SSC, 2% blocking reagent, 0.1% (w/v) N-laurylsarcosine, 50 mmol/L sodium phosphate, pH 7.0) at 68 °C for 1–2 h. Hybridization was carried out overnight at 68 °C in the same buffer with the addition of DIG-labelled PSTVd(−) RNA probe. Then, the membrane was washed twice in 2 × SSC/0.1% SDS for 5 min at room temperature, followed by two more washes in 0.1 × SSC/0.1% SDS for 15 min at 68 °C. The detection was performed with an anti-DIG-antibody (Ab) conjugated to alkaline phosphatase and visualized with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate ready-to-use tablets (Roche).
6
Southern Blot Analysis of DNA
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For DNA analysis, total DNA was extracted using the DNA Blood & Tissue kit (QIAGEN) and quantified using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). For Southern blot analysis, total DNA (10–15 μg) was digested overnight with BamHI mixed with 10× loading dye and separated on an 0.8% agarose gel at 20 mV overnight. The gel was immersed in 0.25 M HCl for 10 min, incubated twice for 15 min in depurination buffer, followed by a 15-min incubation in neutralization buffer. The gel was supported on a layer of Whatman 3MM paper with a tank containing 20× saline sodium citrate (SSC) nucleic acid transfer buffer. A Hybond-XL nylon membrane from Amersham Biosciences was soaked with buffer and placed on top of the gel, taking care to remove any bubbles. Once the paper towel was positioned, a weight was balanced on top, and the apparatus was left overnight to allow the complete transfer. The following day the apparatus was disassembled, and the nylon membrane was exposed to UV radiation for 1 min to cross-link the DNA to the membrane permanently. The GFP gene was used to generate DNA fragments that were labeled with 32P (Prime-It II random primer labeling kit, Agilent Technologies) and used as a probe. The hybridization was performed in Church’s buffer at 65°C for 16 h.
7
Detecting Expanded CRISPR-Edited Repeats
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To determine the presence of the (CTG⋅CAG)500 repeat in CRISPR/Cas9 genome-edited DM500 myoblasts, we used PCR amplification of the (CTG⋅CAG)500 repeat-containing gene segment, followed by Southern blot hybridization. PCR products were resolved by electrophoresis on a 1% agarose gel, transferred by capillary transfer to a Hybond-XL nylon membrane (Amersham Pharmacia Biotech), and hybridized with 32P-labeled oligonucleotides. A DMPK oligo (5′-AGAACTGTCTTCGACTCCGGG-3′), located 5′ of the CRISPR cleavage sites, was used to visualize PCR products from both the unmodified DMPK gene and from cells with a deletion of the region between the two CRISPR sites. The oligo was 5′ end labeled using ɣ32P-ATP and T4 polynucleotide kinase and hybridized to the membrane in Church-Gilbert hybridization solution overnight at 42°C. The membrane was washed, exposed to a Bio-Rad Phosphor Imaging Screen, and imaged by Phosphor-Imager analysis (Molecular Imager FX, Bio-Rad). Analysis was performed with Quantity One (Bio-Rad) and ImageJ software. After imaging, the probe was stripped from the membrane using boiling buffer (0.1 X SSC and 0.1% SDS). Subsequently, using similar conditions for hybridization, washing, and exposure analysis, 32P-end-labeled (CAG)9 probe was used to visualize PCR products containing an expanded (CTG⋅CAG)n repeat.
8
Quantification of Recombinant Protein
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Polyclonal antibodies for TPS4 were generated in rabbits using two synthetic peptides corresponding to residues 58 to 72 and 455 to 469 (Eurogentec, Herstal, Belgium). In order to determine the amount of soluble recombinant protein in the bacterial extracts, 10 µL of crude extract were resolved by SDS PAGE and transferred onto a Hybond-XL nylon membrane (Amersham Biosciences) using a Bio-Rad blotting apparatus with blot buffer (25-mM Tris-HCl, 192-mM glycine, 20% (v/v) methanol). The membrane was blocked with 2% (w/v) bovine serum albumin in TBST (10-mM Tris-HCl (pH 8.0), 150-mM NaCl, 0.05% (v/w) Tween 20) and then incubated first with the polyclonal antiserum (1:500 in TBST) and then with alkaline phosphatase-conjugated antirabbit IgG (Sigma, Deisenhofen, Germany). For visualization, the NBT/BCIP Liquid Substrate system (Sigma) was used.
9
Northern Blot Analysis of SRP RNA
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Total RNA was isolated using TRIzol (Invitrogen) and resolved alongside a single-stranded RNA ladder (New England Biolabs) in a 6% polyacrylamide gel containing 7 M urea. The RNA was transferred to a Hybond-XL nylon membrane (Amersham) and crosslinked by UV irradiation (120 mJ; UV Stratalinker 2400, Stratagene). Crosslinked membranes were blocked with ULTRAhyb-Oligo solution (Ambion) for 30 min at 42°C, then incubated overnight at 47°C with DNA probes complementary to the SRP RNA (Supplementary Table S3), which had been 5′ end radiolabeled according to manufacturer's instructions (KinaseMax, Ambion). Hybridized membranes were washed four times, 30 min each (2x SSC, 0.5% SDS), then imaged using a storage phosphor screen (Amersham).
10
RNA Binding Assay with GST-Tagged Protein
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The indicated amounts of purified GST:p33ΔTMD or GST were incubated with 1 µM of an RNA transcript in 10 µL binding buffer (10 mM HEPES-NaOH pH 8.0, 50 mM KCl, 100 mM EDTA, and 5% glycerol) for 20 min at 28 °C with gentle shaking. Subsequently, the incubated mixture was electrophoresed in 6% non-denaturing PAGE using 1X TAE buffer and transferred onto Hybond™-XL nylon membrane (Amersham Biosciences, Little Chalfont, United Kingdom) via a semi-dry transfer cassette (Bio-Rad Laboratories, Hercules, CA, USA) and was immobilized by a UV-crosslinker (UVP, Upland, CA, USA). The crosslinked membrane was blocked with Denhardt’s solution (VWR, Radnor, PA, USA) for 30 min at 60 °C before adding a DIG-labeled probe. After overnight hybridization, the location of RNA transcripts was detected using DIG-Northern-Detection-system (Roche, Basel, Switzerland) per the manufacturer’s instructions. The Signals were captured by a ChemiDoc™ (Bio-Rad Laboratories, Hercules, CA, USA). The Hill equation was plotted using the default function in Prism version 8 (GraphPad, San Diego, CA, USA).
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