The assay was performed as described previously with some modifications [34 ]. RNA primer 15 (3 μM) and template RNA (TMV PQS5 or TMV PQS5-mut, 6 μM) were annealed in 10 mM phosphate buffer pH 7.0 supplemented with KCl at different concentrations (0.1 mM, 10 mM, 100 mM) by heating at 95°C for 5 min and being cooled down to room temperature slowly. Then 1 μL annealed sample was added into 19 μL reaction buffer (50 mM HEPES pH 7.0, 20 mM NaCl, 5 mM MgCl2 and 4 mM DTT) with 3Dpol (0.2 mg/reaction) and NTPs (200 μM), and the mixture was incubated at 33°C for 60 min. The reactions were stopped by adding an equal volume of 95% formamide and being heated at 90°C for 4 min. The products were separated through electrophoresis on 20% denaturing polyacrylamide gels. The gels were scanned by the Amersham Typhoon 5 (Cytiva, United States) in fluorescence mode. The sequences used in this assay were listed in S3 Table .
Amersham typhoon 5
Manufactured by Cytiva
Sourced in United States
The Amersham Typhoon 5 is a phosphorimager designed for high-resolution imaging of radiolabeled samples, such as protein and nucleic acid gels and blots. It utilizes a photomultiplier tube and a precision laser scanning system to capture digital images of radioactive samples.
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5 protocols using amersham typhoon 5
1
In Vitro RNA Polymerase Assay for TMV PQS
2
Monitoring APC/C-Dependent Ubiquitylation
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Qualitative assays to monitor APC/C-dependent ubiquitylation were performed as previously described [91 (link)]. In brief, reactions were mixed on ice, equilibrated to room temperature before the reactions are initiated with Ub or meUb, and quenched at the indicated time points with SDS. TTF2 ubiquitylation was monitored by mixing 100 nM APC/C, 1 μM Cdh1, 5 μM UBE2C, 5 μM UBE2S (when indicated), 1 μM UBA1, 5 μM TTF2, 5 mM Mg-ATP, and 150 μM Ub or meUb (S2 Fig ). Ubiquitylation of UHRF1 wild type or its variants by APC/C were performed with 100 nM APC/C or pE-APC/C, 1 μM Cdh1 or Cdc20, 0.4 μM UBE2C, 0.4 μM UBE2S (when indicated), 1 μM UBA1, 0.4 μM UHRF1, 5 mM Mg-ATP, and Ub or meUb (Fig 4 and S4 Fig ). Following SDS-PAGE, ubiquitylation products of the fluorescently labeled substrates were resolved by SDS-PAGE and imaged with the Amersham Typhoon 5 (Cytiva Life Sciences, Logan, Utah).
3
DNA Nanoparticle Self-Assembly Protocol
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Self-assembly of DNA sequences of triangular nanoparticles was designed following our previous report [39 (link)]. Equimolar concentrations of individual strands (1 µM) were mixed in presence of various concentrations of conjugated ODNs in TMS buffer. To facilitate hybridization, the solution mixture of DNAs was heated to 80 °C for 5 min and slowly cooled down to 4 °C at a rate of 1 °C/min. An equal volume of loading buffer (sucrose 40% (wt/vol), 0.1% (wt/vol) xylene cyanol, and 0.1% (wt/vol) bromophenol blue) was added to each sample with subsequent analysis on 6% native PAGE in TBM. The gels were run at 60 V for 60 min at room temperature and imaged using ChemiDoc XRS (BioRad, Hercules, CA, USA) or Amersham Typhoon-5 (Cytiva, Marlborough, MA, USA) imaging systems with and without the presence of ethidium bromide.
4
Ubiquitin Conjugation Activity Assay
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A tube with 16.6 µM UBE2R2 and 27 µM K48R or K48R/R74E ubiquitin was diluted in non-reducing reaction buffer (30 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2 and 2 mM ATP); 9 µl of this solution was then aliquoted to autoclaved reaction tubes. A twofold dilution series was generated with E1 protein in non-reducing reaction buffer, with the highest concentration being 10 µM. Reactions were initiated by adding 1 µl of the E1 serial dilution to each tube, followed by brief vortexing. After a 15 min incubation period at room temperature, reactions were quenched in non-reducing 2× SDS–PAGE buffer (100 mM Tris-HCl pH 6.8, 20% glycerol, 30 mM EDTA and 4% SDS). Products were separated by SDS–PAGE on 18% Tris-Glycine gels and then stained with Coomassie blue solution (20% methanol, 10% acetic acid and 0.1% Coomassie blue). The experiment was then repeated using 32P-labeled donor ubiquitins before exposure to a phosphor screen. Substrates and products were visualized by scanning on an Amersham Typhoon 5 (Cytiva) and quantified using ImageQuant software v.8.2.0.0 (GE Healthcare). The fraction of product signal was determined by dividing the UBE2R2~donor ubiquitin signal by the total signal in the lane (Extended Data Fig. 4e ). Experiments were performed in duplicate technical replicates.
5
Native Equine Protein Purification and 2D-Gel Electrophoresis
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Native Equ c 1 purified from the Breed/Gender Mix horse hair extract was prepared (2‐D Clean‐Up Kit, GE Healthcare) for two‐dimensional (2D)‐gel electrophoresis that was performed with immobilized pH gradient strips using the PROTEAN i12 isoelectric focusing system (Bio‐Rad Laboratories) as previously described.
17 (link) Gels were stained with SYPRO Ruby protein gel stain (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions. Images were recorded using the Amersham typhoon gel and blot imaging system (Amersham Typhoon 5, Cytiva).
17 (link) Gels were stained with SYPRO Ruby protein gel stain (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions. Images were recorded using the Amersham typhoon gel and blot imaging system (Amersham Typhoon 5, Cytiva).
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