The standard helix destabilizing assay was performed as previously described [40 (link)] with minor modifications. Briefly, 20 pmol of recombinant protein and 0.1 pmol of HEX-labeled helix substrate were added to a mixture containing 50 mM HEPES-KOH (pH 7.5), 2.5 mM MgCl2, and 2 mM Dithiothreitol (DTT), 0.01% bovine serum albumin (BSA), and 15 U RNasin (Promega). After incubation at 37°C for 60 min or indicated time, the reaction was terminated by adding proteinase K (final concentration of 1 μg/μl) and 5×loading buffer [100 mM Tris-HCl, 1% SDS, 50% glycerol, and bromophenol blue (pH 7.5)]. The mixtures were then electrophoresed on 15% native-PAGE gels, followed by scanning with a Typhoon 9200 imager (GE Healthcare, Piscataway, NJ). The RNA strand hybridization assay was performed as previously described [40 (link)]. The sequences of the stem-loop-structured RNA strands were indicated in Fig 7A . The samples were also resolved on 15% native-PAGE gels, followed by gel scanning a Typhoon 9200 imager (GE Healthcare).
Typhoon 9200 imager
Manufactured by GE Healthcare
Sourced in United Kingdom
The Typhoon 9200 imager is a high-performance instrument designed for fast and sensitive detection of fluorescent and chemiluminescent signals in a variety of life science applications. It utilizes laser-based excitation and sensitive photomultiplier tube (PMT) detection to capture digital images of protein and nucleic acid gels, microarrays, and other samples.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Imagequant software, by GE Healthcare (3 mentions)
Hybond n membrane, by GE Healthcare (2 mentions)
Decyder software, by GE Healthcare (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bio lyte 3 10, by Bio-Rad (1 mentions)
Multiphor 2 electrophoresis system, by GE Healthcare (1 mentions)
Biolyte 4 6, by Bio-Rad (1 mentions)
Proteinase k, by New England Biolabs (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Prime a gene labeling system, by Promega (1 mentions)
Rnasin, by Promega (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
Dylight 633 maleimide, by Thermo Fisher Scientific (1 mentions)
Sybr green 1 nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Endo h, by New England Biolabs (1 mentions)
Anti flag, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Hybond n membrane, by Cytiva (1 mentions)
22 protocols using typhoon 9200 imager
1
Helix Destabilization and RNA Strand Hybridization Assay
2
Northern Blot Analysis of Viral RNA
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At 72 h p.t., intracellular RNA was extracted using TRizol reagent (Invitrogen) according to the manufacturer's instructions. To analyze viral RNAs by Northern blot hybridization, 10 μg total RNA was denatured for 10 min at 65 °C in loading buffer (50% deionized formamide, 18% formaldehyde, 1x MOPS) and separated in a 1% (w/v) agarose and 2.2 M formaldehyde-containing, 1x MOPS-buffered gel at 16 V for 16–17 h. The gel was soaked in buffer A (50 mM NaOH, 150 mM NaCl) for 30 min and then in buffer B (100 mM Tris-HCl / pH 7.5, 150 mM NaCl) for 30 min. Next, the RNA was transferred onto a positively charged nylon membrane by vacuum blotting. The RNA was cross-linked to the membrane and hybridized with an [α-32P]dCTP-labeled DNA probe specific for HCoV-229E nucleotides 26857–27277 and the negative-strand complement of this sequence (TaKaRa Bio Inc). Following hybridization, membranes were rinsed 2 times with 2x SSC/0.01% (w/v) SDS at room temperature and 2 times with 0.2x SSC/0.01% (w/v) SDS at 55 °C for 30 mins. Hybridization signals were visualized by autoradiography using a Typhoon 9200 imager (GE Healthcare).
3
In Vitro Activity Assays of Coronavirus nsp8
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For in vitro activity assays, the various forms of coronavirus nsp8 produced in this study were incubated with single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), and partial-duplex substrate RNAs. In addition, a range of 3′-biotinylated substrate RNAs was used. Unless stated otherwise in the text, the reaction mixtures contained 50 mM Tris-Cl, pH 8.0, 50 mM NaCl, 1 MgCl2, 1% Triton X-100, 1 mM DTT, 1.5 mM β-mercaptoethanol, 4.5% glycerol, 1 μM substrate RNA, 100 μM the indicated NTP(s), 0.17 μM the indicated [α-32P]NTP(s), and 2 μM nsp8. The reaction mixtures were incubated for 60 min at 30°C, and the reactions were terminated by the addition of sodium acetate (pH 5.2; final concentration, 300 mM) and 10 volumes of ice-cold ethanol. Following centrifugation, the air-dried pellets were resuspended in PCR-grade proteinase K solution (final concentration, 1 mg/ml; Invitrogen) and incubated at 55°C for 15 min. Reactions were stopped by adding Fu-mix (6 M urea, 80% deionized formamide, 1× TBE [Tris-borate-EDTA], 0.1% [wt/vol] bromophenol blue, 0.1% [wt/vol] xylene cyanol). Following denaturation for 10 min at 65°C, the reaction products were separated in 1× TBE-buffered 12% polyacrylamide gels containing 7 M urea and analyzed by phosphorimaging using a Typhoon 9200 imager (GE Healthcare) and Quantity One software (Bio-Rad).
4
RNA Expression Analysis by Northern Blot
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To confirm the expression and size of putative transcripts total RNA from GC was separated on a denaturing 15% polyacrylamide gel containing 8 M urea. Electrophoresis was performed in 1× TBE buffer at 150 V for ∼3 h. After transferring the RNA onto a nylon membrane by wet blotting in 0.5× TBE at 25 V for 1 h, it was covalently cross-linked by ultraviolet irradiation. For the specific detection of putative sRNAs 24-mer DNA oligonucleotides antisense to the RNAs were end labeled with (γ32P)-adenosine triphosphate (ATP). Membranes were hybridized with the respective probes (Supplementary Table S5) at 42°C for 6 h in hybridization buffer (Rapid-Hyb, GE Healthcare) and washed twice with washing buffer (2× SSC, 0.1% sodium dodecyl sulphate (SDS)) at 45°C. Blots were exposed to phosphor storage screens (Fujifilm) which were then scanned by Typhoon 9200 imager (GE Healthcare).
5
Quantifying Peptide-Labelled PapMV-SrtA Nanoparticles
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Peptide-labelled PapMV-SrtA nanoparticles were diluted to 0.1 µg/µL in cathode buffer for Tris-Tricine gels, or in Tris–Glycine migration buffer supplemented with 30% of SDS loading buffer (50% glycerol, 2% SDS, 0.002% bromophenol blue, 14% 2-mercaptoethanol). Samples were then heated at 95 °C for 10 min and 4 µL of solution (0.4 µg) was loaded on 10% Tris-Tricine SDS-PAGE for the gels shown in Fig. 4 or 15% Tris/Glycine SDS-Page for the gels shown in Fig. 3 and Additional file 2 : Figure S2. Gels were colored with Sypro-Ruby gel stain (Life Technologies Inc. Carlsbad, California, USA) following the manufacturer’s rapid staining protocol. Sypro-Ruby fluorescence was detected with the 610 nm emission filter following excitation using the green laser (532 nm) of the Typhoon 9200 imager (GE Healthcare Life Sciences). The fluorescence signal resulting from protein bands was quantified using the image analysis software ImageQuant 5.2, and the intensity volumes associated to the different bands, corrected for background signal, were used for the conjugation quantification analysis. The conjugation efficiency was calculated by dividing the signal of coupled PapMV-SrtA protein band by the sum of conjugated and unconjugated PapMV-SrtA signals.
6
Two-Dimensional Gel Electrophoresis of Proteins
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Samples of PMS (75 μg) or m7GTP-Sepharose of each experimental condition were added to 8.5 M urea/5% β-mercaptoethanol (Bio-Rad) and loaded into horizontal IEF slab gels as the first dimension. IEF was performed with immobilized pH 3‒10 nonlinear gradient (3% v/v Bio-Lyte 4/6, 2% v/v Bio-Lyte 3/10) (Bio-Rad) strips (10 cm) in a flatbed Multiphor II Electrophoresis System (GE Healthcare), according to the manufacturer’s instructions. The first dimension was combined with standard vertical slab SDS-PAGE as the second dimension (12% acrylamide; 2.6% cross-linking) (GE Healthcare) performed in 1.0 mm thick gels with the IEF strip used as stacking gel, as described previously [12 (link), 17 (link), 18 (link)]; and then proteins identified by western blot. Protein markers (see above) and pI standards (range: 3–10) (GE Healthcare) were used to calculate the apparent MW and pI of identified proteins.
In other experiments, immunoprecipitated samples were labeled on their lysine residues with Cy3 and Cy5 minimal dyes (GE Healthcare). Pairs of experimental and control samples were mixed and analyzed by 2-DGE as described above. The fluorescent gels were scanned using a Typhoon 9200 imager (GE Healthcare).
In other experiments, immunoprecipitated samples were labeled on their lysine residues with Cy3 and Cy5 minimal dyes (GE Healthcare). Pairs of experimental and control samples were mixed and analyzed by 2-DGE as described above. The fluorescent gels were scanned using a Typhoon 9200 imager (GE Healthcare).
7
In Vitro Ribozyme Assay Protocol
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The ribozyme RNA was synthesized from the in vitro transcription using T7 RNA polymerase, and the 5′-HEX-labeled substrate RNA was synthesized by TaKaRa. Indicated amount of protein was added in 10 μl reaction volumes containing 0.15 nM substrate, 0.3 nM ribozyme, 50 mM HEPES-KOH (pH 7.5), 2.5 mM MgCl2, and 2 mM DTT, 0.01% BSA, 20 U RNasin. The mixtures were incubated at 37°C for 30 min, and then treated by proteinase K (final concentration of 1 μg/μl) at 37°C for 15 min. The digestion reactions were precipitated with 20 μl isopropanol and 2 μg glycogen (-80°C, 30 min), and subjected to by centrifugation at 12 000 g for 15 min. The precipitates were dissolved in formamide and electrophoresed on 15% acrylamide-7 M urea gels, followed by scanning with a Typhoon 9200 imager (GE Healthcare).
8
RNA Helix-Destabilizing Assays with σNS
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RNA helix-destabilizing assays were conducted as described38 (link) with modifications. Briefly, various concentrations of σNS, σNS-R6A, and σNS-ΔN17 (2.5, 5, 10, and 20 µM) and 0.5 μM dsRNA helix substrate were incubated at 37 °C for 1 h in a reaction volume of 20 μL containing 25 mM HEPES-KOH (pH 8.0), 100 mM NaCl, 2 mM MgCl2, 2 mM DTT, and 5 U RNase-OUT (Invitrogen). Reactions were terminated by the addition of 5 U proteinase K (NEB) for 15 min and 2.5 μL 5× loading buffer (100 mM Tris-HCl (pH 7.5), 50% glycerol, and bromophenol blue). The samples were electrophoresed in 4–20% native polyacrylamide gels (Genscript USA), and gels were scanned using a Typhoon 9200 imager (GE Healthcare). Bacterial-expressed maltose binding protein (MBP; 20 μM) and norovirus p41 protein (20 μM) were used as negative and positive controls, respectively. The effect of bile acids on σNS RNA helix-destabilizing activity was determined by incubating 20 and 40 μM of different components of bile acid derivatives, including TASAH, SGAH, CHAPS, and CHAPSO, with 20 μM σNS for 1 h before initiation of the helix-destabilization assay. Experiments were conducted three times.
9
Kinase Assay for Gh-LYK1-ID and Gh-LYK2-ID
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For the kinase assays, the purified Gh-LYK1-ID or Gh-LYK2-ID protein was individually incubated with γ-32P ATP at 30°C for 30 min, as previously described (Liu et al., 2011 (link)). The phosphorylated substrate was visualized with a Typhoon 9200 imager (GE Healthcare) after separation through SDS-PAGE.
10
Mapping CENP-A Nucleosome Structure
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CENP-A nucleosomes assembled with HEX-labelled 147 bp α-satellite DNA20 (link) were reconstituted and then purified using a sucrose gradient. An amount of 4 μg of HEX-labelled CENP-A nucleosomes alone or complexed to CENP-NNT were used in each reaction. The hydroxyl radical cleavage reaction was initiated by addition of 5 μl of 40 mM FeAmSO4/80 mM EDTA, 2 M ascorbate and 2.4% H2O2 to a 30 μl reaction mixture. Each reaction was carried out for 5 min at room temperature, and terminated with 200 μl of stop solution (0.1% SDS, 25 mM EDTA, 1% glycerol and 100 mM Tris, pH 7.4). Further phenol/chloroform extraction and ethanol precipitation was carried out to extract DNA fragments. Samples were separated by denaturing PAGE (10% polyacrylamide, 7 M urea, 88 mM Tris-borate and 2 mM EDTA, pH 8.3)20 (link). Gels were imaged on a Typhoon 9200 imager (GE Healthcare). Band intensities were quantified from ImageJ from three independent experiments.
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