For the analysis of de-novo protein synthesis, we collect equal number of radiolabeled cells and prepared two aliquots with equal volumes of shCTR and shWRN cells extracts for analyses by 10% PAA gels. One of the PAA gels was used for Coomassie Brilliant Blue (CBB) staining (loading control) while the second was used for 35 S-met/cys measurement. Both gels were vacuum-dried, exposed to a phosphoscreen and developed using a Pharos FX molecular imager (BIO-RAD). The data were plotted using Graph Pad Prism (GraphPad Prism, RRID:SCR_002798) and the signi cance determine by Student´s t-test of three independent samples. Immunoprecipitation of 35 S-met/cys labeled proteins Extracts derived from 35 S-met/cys labeled cells samples were subjected to immunoprecipitation assay. To this end, we used the same concentration of speci c antibodies against G6PD, Ku70 and Tubulin. Reactions were incubated overnight at 4 °C in a roller and the immunocomplexes were captured using Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, sc-2003,) following the manufacturer's instructions. The immunoprecipitated complexes were resolved in 10% PAA and stained with CBB followed by vacuum drying. Lastly, the gels were scanned to visualize IgG and then exposed to Phosphoscreen and imaged using a Pharos FX molecular imager (BIO-RAD).
Pharos fx molecular imager
Manufactured by Bio-Rad
Sourced in United States, Japan
The Pharos FX Molecular Imager is a lab equipment designed for the detection and analysis of fluorescent and chemiluminescent signals in various applications, such as Western blotting, gel imaging, and microplate analysis. The device uses high-performance optics and sensitive detectors to capture detailed images of samples, enabling researchers to visualize and quantify target molecules with accuracy.
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Lab products found in correlation
Quantity one software, by Bio-Rad (5 mentions)
Transcriptaid t7 high yield transcription kit, by Thermo Fisher Scientific (2 mentions)
T7 endonuclease 1, by New England Biolabs (2 mentions)
Dna clean concentrator 5 kit, by Zymo Research (2 mentions)
Super gelred, by US Everbright (2 mentions)
P 6 gel columns, by Thermo Fisher Scientific (2 mentions)
Novex tbe urea gel, by Thermo Fisher Scientific (2 mentions)
Locally averaged background correction, by Bio-Rad (2 mentions)
Semi dry transfer cell, by Bio-Rad (2 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Urea page gel, by Americanbio (1 mentions)
Scion image software, by Techcomp Instruments (1 mentions)
Cas9 nuclease, by New England Biolabs (1 mentions)
Rnase 1, by Thermo Fisher Scientific (1 mentions)
Rnase t1, by Thermo Fisher Scientific (1 mentions)
T4 dna polymerase, by New England Biolabs (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
810 spectropolarimeter, by Jasco (1 mentions)
Pegfp c1 vector, by Takara Bio (1 mentions)
42 protocols using pharos fx molecular imager
1
De-novo Protein Synthesis Analysis
2
De-novo Protein Synthesis Analysis
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For the analysis of de-novo protein synthesis, we collect equal number of radiolabeled cells and prepared two aliquots with equal volumes of shCTR and shWRN cells extracts for analyses by 10% PAA gels. One of the PAA gels was used for Coomassie Brilliant Blue (CBB) staining (loading control) while the second was used for 35 S-met/cys measurement. Both gels were vacuum-dried, exposed to a phosphoscreen and developed using a Pharos FX molecular imager (BIO-RAD). The data were plotted using Graph Pad Prism (GraphPad Prism, RRID:SCR_002798) and the signi cance determine by Student´s t-test of three independent samples. Immunoprecipitation of 35 S-met/cys labeled proteins Extracts derived from 35 S-met/cys labeled cells samples were subjected to immunoprecipitation assay. To this end, we used the same concentration of speci c antibodies against G6PD, Ku70 and Tubulin. Reactions were incubated overnight at 4 °C in a roller and the immunocomplexes were captured using Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, sc-2003,) following the manufacturer's instructions. The immunoprecipitated complexes were resolved in 10% PAA and stained with CBB followed by vacuum drying. Lastly, the gels were scanned to visualize IgG and then exposed to Phosphoscreen and imaged using a Pharos FX molecular imager (BIO-RAD).
3
In Vitro RNA Polymerase Assay
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3Dpol was a gift from P. Gong (Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China). The assay was performed as described previously (40 (link)). P15 (300 nM) and template RNA (600 nM) were annealed in a reaction buffer [50 mM Hepes, 20 mM NaCl, 1.0 mM KCl, 5 mM MgCl2, and 4 mM DTT (pH 7.0) at 25°C] after heating at 85°C for 2.0 min and slowly cooled to 4°C. Subsequently, NTPs (final concentration, 200 μM) and 3Dpol (0.02 mg/reaction) were added, and the reactions were performed at 33°C for 20 min. The reactions were stopped after adding an equal volume of stop buffer (95% formamide) and heated at 90°C for 4.0 min. The products were loaded and separated on 20% denaturing polyacrylamide gels. Finally, the gels were scanned by a Pharos FX Molecular Imager (Bio-Rad) operated in fluorescence mode.
4
Single Nucleotide RT Incorporation Assay for Cellular dNTP Detection
5
Quantitative Mutation Detection by ACB-PCR
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Following ACB‐PCR, 10 µl of 6× ficoll loading buffer/dye were added to each 50 µl reaction, and 10 µl of each ACB‐PCR product were analyzed on 8% nondenaturing, polyacrylamide gels. A PharosFX Molecular Imager with an external blue laser (Bio‐Rad) was used to visualize the fluorescent ACB‐PCR products. Quantity One software (Bio‐Rad), with a locally averaged background correction, was used to quantify the pixel intensities of the correct‐sized bands. For the PIK3CA E545K, log‐log plots relating MF to fluorescence were constructed and fit with a power function. For the PIK3CA H1047R, KRAS G12D, and G12V mutations, log‐linear plots relating MF to fluorescence were constructed and fit with a logarithmic function. Using the function of the standard curve and the pixel intensities of the ACB‐PCR products, the MF (ratio of mutant to wild‐type sequence) of each unknown sample was calculated.
6
Transcriptional Regulation Profiling
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Nuclear extracts were prepared as described [67 (link)]. EMSA was performed as described [68 (link)] using various P32-labeled DNA probes (Supplementary Table 1 ) specific for mouse IL-10 or HAVCR2 promoter. Bands were visualized using a phosphoimager (PharosFX Molecular Imager, Bio-Rad). Immunoblot analysis was carried out as described [66 (link)]. The pull-down of DC nuclear proteins using streptavidin (SA)-coupled Dynabeads and indicated biotinylated oligonucleotides (see Supplementary Table 1 ) followed by immunoblot analysis was performed as illustrated previously [69 (link)]. Densitometry quantification was done using Scion Image software (Scion Corporation, Maryland, USA).
7
Diverse Enzymatic Toolkit for Molecular Biotechnology
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T4 DNA polymerase, Cas9 Nuclease, Streptococcus pyogenes (Cas9), Bst DNA Polymerase (Bst DNA pol), M‐MuLV Reverse Transcriptase (M‐MuLV RT), and T7 Endonuclease I were commercially available from New England Biolabs (USA). Oligonucleotide sequences containing primers are provided in Table S1 in the Supporting Information. Transcript Aid T7 High Yield Transcription kit, RNase I, and RNase T1 were purchased from Thermo Fisher Scientific. TaKaRa Shuzo Co. Ltd. (Tokyo, Japan) provided Pyrobest DNA Polymerase and PrimeSTAR HS DNA Polymerase, which were mainly used to generate target DNA. Zymo Research Corp provided DNA Clean & Concentrator‐5 kit. The nucleic acid stains Super GelRed was purchased from US Everbright Inc. (Suzhou, China). Gel Imaging was performed on Pharos FX Molecular imager and ChemiDoc MP Imaging System (Bio‐Rad, USA). The Tm measurements were executed on a JASCO‐810 spectropolarimeter (JASCO, Easton, MD, USA) equipped with a Peltier temperature controller.
8
Primer Extension Assay for HIV-1 RT Activity
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An HIV-1 RT primer extension assay was performed as previously described with slight modifications [53 (link)]. A 5′ 32P-labeled 17-mer DNA primer (5′-CGCGCCGAATTCCCGCT-3′) was annealed to a 40-mer RNA template (5′-AAGCUUGGCUGCAGAAUAUUGCUAGCGGGAAUUCGGCGCG-3′) in the presence of 100 mM NaCl, 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA. 20 µL Reaction mixtures contained 10 nM template-primer, 4 µL of purified HIV-1 RT, 5 µM or 50 nM of all four dNTPs (ThermoScientific), 12.5 mM Tris–HCl (pH 7.5), 12.5 mM NaCl, 2.5 mM MgCl2 and 20 µM oligo(dT). Reactions were initiated upon addition of RT and incubated at 37 °C for 1 h (for Fig. 3 b) and 5 min (for Fig. 3 d). Reactions were terminated with 10 µL of 40 mM EDTA, 99 % formamide and the products were resolved on a 14 % urea-PAGE gel (American Bio Sequel NE reagent) and imaged using Pharos FX molecular imager (Biorad) and analyzed using ImageLab software.
9
Single Nucleotide Extension Assay for HIV-1 Reverse Transcriptase
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This single nucleotide extension assay was modified from a previously described assay [51 (link)]. A 5′ 32P-labeled DNA 18-mer (5′-GTCCCTCTTCGGGCGCCA-3′) was annealed to a 19-mer DNA template (3′-CAGGGAGAAGCCCGCGGTG-5′) at a 1:2 ratio. Extension from an 18-mer to 19-mer indicates that clofarabine triphosphate has been incorporated by HIV-1 reverse transcriptase. 20 μL reactions contained 200 fmol template/primer, 2 μL clofarabine triphosphate at concentrations indicated or 50 μM of dNTPs for the positive control, 4 μL of purified RT (HIV-1 NL4-3), 25 mM Tris–HCl, pH 8.0, 2 mM dithiothreitol, 100 mM KCl, 5 mM MgCl2, and 10 µM oligo(dT). Reactions were incubated at 37 °C for 5 min and then quenched with 10 µL of 40 mM EDTA and 99 % (vol/vol) formamide at 95 °C for 2 min. The reactions were resolved on a 20 % urea-PAGE gel (American Bio Sequel NE reagent) and imaged using Pharos FX molecular imager (Biorad).
10
Fluorescent RNA Hybridization and Analysis
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