Electrophoresis mobility shift assays (EMSA) were performed using a 120bp (2684–46 nt of circular genome) of the intergenic region (IR) of ChiLCV as a probe. 100ng of double-stranded oligonucleotides were labelled with a Klenow fragment (Thermo Scientific, Massachusetts, USA). EMSA reactions were carried out at 25° C for 60 min in 80 μL of buffer (10 mM Hepes-KOH, pH 8.0, 100 mM KCl, 5mM MgCl2 , 30mM NaCl, 1 mM DTT, and 20% glycerol). The EMSA reaction products were loaded onto an 8% polyacrylamide gel and the gel was allowed to run at 120V for 3 h. Further, the gel was dried and kept overnight in radiation-sensitive plates and the signal was detected on a phosphorimager (Typhoon 9200, Amersham, Little Chalfont, UK).
Typhoon 9200
Manufactured by Cytiva
Sourced in United Kingdom, United States
The Typhoon 9200 is a versatile fluorescence and phosphorimaging system designed for a wide range of applications. It can detect and quantify a variety of fluorescent and radioactive labels in gels, blots, and microarrays. The system utilizes a flexible optical design and multiple excitation sources to provide high sensitivity and dynamic range.
Automatically generated - may contain errors
Lab products found in correlation
Klenow fragment, by Thermo Fisher Scientific (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Mouse anti pdi, by Abcam (1 mentions)
Mouse anti gfp, by Fortrea (1 mentions)
Superase in, by Thermo Fisher Scientific (1 mentions)
Rna clean concentrator 5 kit, by Zymo Research (1 mentions)
Nuclease p1, by US Biological (1 mentions)
4 protocols using typhoon 9200
1
Electrophoretic Mobility Shift Assay for ChiLCV Intergenic Region
2
Fluorescent Visualization of UNG Activity
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UNG activity was measured using 40-mer oligodeoxynucleotide duplex containing a single U:A mispair as described.12 (link) Products were resolved by denaturing polyacrylamide gels (20%), prior to visualization with a Typhoon 9200 fluorescence imager (Amersham Bioscience via GE Healthcare, Pittsburgh, PA, USA) and UNG activity was calculated (percentage of cut band relative to total oligonucleotide).
3
Comprehensive Protein and RNA Analysis
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Total yeast RNA isolation by the hot phenol method, Northern blot analysis, total protein extractions and Western blot analysis, and anti-1a, anti-2apol, anti-Dpm1, anti-FLAG and anti-Pgk1 antibodies were as described [4 , 15 (link), 81 (link), 82 (link)]. Mouse anti-PDI was acquired from Abcam, rabbit and mouse anti-HA antibodies were purchased from Santa Cruz and Roche, respectively. Mouse anti-GFP was purchased from Covance. Northern blots were imaged on a Typhoon 9200 (Amersham Biosciences, Piscataway, NJ). Band intensities were analyzed by using ImageQuant software (Molecular Dynamics, Piscataway, NJ).
4
Methylation of GpppRNA by Nuclear/Cytoplasmic Extracts
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Reactions were prepared, each containing 5 fmol radiolabeled GpppRNA, 50 mM Tris pH 8.0, 6 mM KCl, 1.25 mM MgCl2, 100 nM S-adenosylmethionine, 2 mM DTT and 20 U SUPERase-In (Thermo Fisher AM2696). Each reaction volume was 10 μl and contained either 4 μg (Figure 1B ) or 1 μg (Figures 1D , 4E and 5C ) nuclear or cytoplasmic extract, 1 μl immunoprecipitated (5% of total) bead slurry (Figure 1B ), or 4 μl gel filtration fraction (Figure 2B ). Reactions were incubated for 30 min at 37°C, and RNA was then immediately purified using the RNA Clean & Concentrator-5 kit (Zymo Research), eluting with 7 μl RNase-free water. Purified RNA was digested in a 7-μl reaction containing 5 μl purified RNA, 50 mM sodium acetate pH 5.2 and 0.44 U nuclease P1 (US Biological N7000). After incubating for 30 min at 37°C, 2 μl of each reaction was spotted on the origin of a pre-run PEI cellulose thin-layer chromatography plate (Macherey-Nagel 801053), which was then developed in 0.4 M ammonium sulfate. After overnight exposure to a storage phosphor screen, images were obtained with a Typhoon 9200 (Amersham).
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