Purified cyclases were mixed at a final concentration of 5 μM with 100 μM NTPs and trace radiolabeled α−32P-NTP in 10 μL reactions with 100 mM KCl, 10 mM MgCl2, 1 mM MnCl2, and 50 mM Tris-HCl pH 8.5. These reactions were incubated for 2 h at 37°C and then terminated by treatment with 1 μl of 5 units μl−1 Calf Intestinal Phosphatase (CIP, New England Biolabs) for 1 h at 37°C. 1 μl of each reaction was spotted on glass-backed silica-coated thin layer chromatography (TLC) plates (Millipore) and developed for one hour in a solvent system composed of 11:7:2 1-propanol:conc. NH4OH:water as previously described (Whiteley et al., 2019 (link)). TLC plates were dried and then processed by exposing with a phosphor screen followed by imaging with an Amersham Typhoon IP (Cytiva). For optimization experiments reactions were left at 37°C for 25 min then terminated at 85°C for 2 min, and CIP treated for 30 min at 37°C before spotting on TLC.
Amersham typhoon ip
Manufactured by Cytiva
Sourced in United States
The Amersham Typhoon IP is a highly sensitive phosphorimager designed for the detection and quantification of radioactively labeled biomolecules, such as proteins and nucleic acids, in gel-based and blotting applications. It utilizes a scanning laser and photomultiplier tube technology to capture high-resolution digital images of radioactive samples.
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6 protocols using amersham typhoon ip
1
Cyclic Nucleotide Synthesis Assay
2
SARS-CoV-2 RdRp Complex Activity Assay
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SARS-CoV-2 RdRp complex was prepared as described previously [15 (link)]. SARS-CoV-2 RdRp activity was measured using a reaction mixture consisting of TRIS-HCl (25 mM, pH8), NTPs (50 mM ATP, CTP and TTP; 25 mM GTP), [α-32P]-GTP (0.1 μM), RNA primer, RNA template, SARS-CoV-2 RdRp complex (0.1 μM) and various concentrations of each compound of interest. After incubating the reaction mixtures at 30 °C for 10 min, MnCl2 (5 mM) was added into each reaction. The RNA polymerase reactions were stopped after 30 min of incubation at 30 °C, by adding formamide containing 40 mM EDTA and heated at 95 °C for 10 min. Then the reaction mixture was resolved on 20% polyacrylamide-urea denaturing gels (SequaGel, National Diagnostics, Atlanta, GA, USA), and the resulting primer extended products were visualized using the Amersham Typhoon IP (Cytiva, Marlborough, MA, USA) and analyzed using the ImageQuant TL 8.2 (Cytiva) [18 (link)].
3
SARS-CoV-2 RdRp Complex Inhibition Assay
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SARS-CoV-2 RdRp complex (nsp12/nsp7/nsp8) was prepared as published [9 (link)]. The RNA synthesis activity of the RdRp complex was evaluated in a reaction mixture comprising a 19-mer RNA primer, 43-mer RNA template, 25 mM TRIS-HCl (pH8), cold NTPs (50 µM ATP, CTP and UTP; 25 µM GTP), 0.1 μM [α-32P]-GTP and different concentrations of each inhibitor of interest. Nuclease-free water was added in place of the RdRp or the inhibitors for the negative control (−Pol) or no-treatment control (+Pol), respectively. After 10 min incubation at 30 °C, 5mM MnCl2 was added into each reaction mix to initiate the RdRp reaction. After another 30 min incubation at 30 °C, the RdRp reactions were terminated with formamide containing 40 mM EDTA, and were heated at 95 °C for 10 min. The resulting reaction products were resolved on 20% polyacrylamide-urea denaturing gels (SequaGel, National Diagnostics, Atlanta, GA, USA) and visualized using the Amersham Typhoon IP (Cytiva, Marlborough, MA, USA). Analyses were subsequently conducted with ImageQuant TL 8.2 (Cytiva) [6 (link)].
4
In vitro Protein Synthesis Assay
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In vitro transcription–translation reactions were carried out using the NEB PURExpress system and its published protocol (https://www.neb.com/protocols/0001/01/01/protein-synthesis-reaction-using-purexpress-e6800 ). Briefly, Solution A, Solution B, and 35S-methionine were combined with 250 ng pAB87, pAB101, or pDHFR as a positive control. The reactions were incubated for 1 h at 37 °C, and 5 µL of the reaction was combined with equal volume Laemalli Sample Buffer containing 10% β-mercaptoethanol to stop the reaction. Chloramphenicol (25 µg/mL final) was added to the transcript–translation reaction after 60 min, to inhibit protein synthesis and samples were mixed with Laemelli Sample buffer at the indicated times. Proteins were separated by SDS-PAGE using a 17.5% polyacrylamide gel, the gel was dried, exposed to a phosphor screen, and imaged with an Amersham Typhoon IP (Cytiva). Samples were also visualized by immunoblot using anti-GFP antibodies as described above.
5
Thermal, Structural Characterization of Samples
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Transition temperatures and enthalpies were recorded with a Q1000 apparatus from TA Instruments, Paris, France. The instrument was calibrated with indium and otherwise stated three heating/cooling cycles were performed on each sample, with a heating and cooling rate of 10 °C/min. SAXS/WAXS patterns were obtained with a transmission Guinier-like geometry. A linear focalized monochromatic Cu Kα1 beam (λ = 1.5405 Å) was obtained using a sealed-tube generator (600 W) equipped with a bent quartz monochromator. In all cases, samples were filled in home-made sealed cells of 1 mm path. The patterns were recorded with a curved Inel CPS120 counter gas-filled detector, Paris, France, linked to a data acquisition computer (periodicities up to 90 Å) and on image plates scanned by Amersham Typhoon IP, Cytiva, France with 25 μm resolution (periodicities up to 120 Å).
6
In vitro Protein Synthesis and Detection
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In vitro transcription-translation reactions were carried out using the NEB PURExpress system and its published protocol (https://www.neb.com/protocols/0001/01/01/protein-synthesis-reaction-using-purexpress-e6800 ). Briefly, Solution A, Solution B, and 35S-methionine were combined with 250 ng pAB87, pAB101, or pDHFR as a positive control. The reactions were incubated for 1 hour at 37 °C and 5 μL of the reaction was combined with equal volume Laemalli Sample Buffer containing 10% β-mercaptoethanol to stop the reaction. Chloramphenicol (25 μg/mL final) was added to the transcript-translation reaction after 60 min, to inhibit protein synthesis and samples were mixed with Laemelli Sample buffer at the indicated times. Proteins were separated by SDS-PAGE using a 17.5% polyacrylamide gel, the gel was dried, exposed to a phosphor screen, and imaged with an Amersham Typhoon IP (Cytiva). Samples were also visualized by immunoblot using anti-GFP antibodies as described above.
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