Phosphorimager screen

Manufactured by GE Healthcare

Sourced in United States, United Kingdom

The PhosphorImager screen is a laboratory equipment used for the detection and quantification of radioactively labeled samples. It functions by capturing the luminescence emitted from phosphor-coated screens that have been exposed to radioactive samples, allowing for the visualization and analysis of these samples.

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Lab products found in correlation

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PhosphorImager (289 citations)

83 protocols using phosphorimager screen

Quantifying m1G37 Modification in tRNA

Cited 1 time

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The extent of RT arrest at m¹G37 in tRNA-Leu-UAA and tRNA-Pro-UGG from S. cerevisiae was quantified via primer extension with AMV RT, an enzyme with low processivity at this modification (Werner et al., 2020 (link)). The primers were designed to enable a 4-nucleotide extension to m¹G37 (tRNA-Leu-UAA: 5′-CGCGGACAACCGTCCAAC-3′; tRNA-Pro-UGG: 5′-TGAACCCAGGGCCTCT-3′) and 5'-end-labeled with γ-³²P-ATP. 3 μg of total RNA from exponentially growing yeast cells was mixed with 1 pmol end-labeled primer and incubated at 95°C for 3 min followed by slow cooling to 37°C. RT reactions were assembled by adding 15 U AMV RT (Promega), 0.5 mM dNTPs, 20 U SUPERase In (Ambion) and 1X AMV RT buffer in a 5-μl volume. Following incubation at 37°C for 45 min, reactions were stopped by addition of 5 μl 2X RNA loading dye (47.5% Formamide, 0.01% SDS, 0.01% bromophenol blue, 0.005% Xylene Cyanol, 0.5 mM EDTA), boiled at 95°C for 5 min, and resolved on a denaturing 15% PAA/7M urea/1X TBE gel. The gel was exposed at −80°C to a PhosphorImager screen, which was scanned on a Typhoon FLA 9000 (GE Healthcare). Band intensity was quantified with ImageQuant (GE Healthcare).

Behrens A., Rodschinka G, & Nedialkova D.D. (2021). High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq. Molecular Cell, 81(8), 1802-1815.e7.

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In Vitro RV Protein Translation Assay

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For the in vitro RV translation assay, various concentration of ORNs RV S1 AUG (0.5, 1, 2 or 4 µM), S10.2, S11.2 (1 or 4 µM) or Scr (4 µM) were incubated with 300 ng of S1, S10 or S11 RV transcripts purified by phenol-chloroform-isoamyl alcohol, followed by exclusion chromatography (Illustra Microspin G-25 column, GE Healthcare, Chicago, IL, USA) to remove other impurities. The mixtures of RNA and ORN were incubated for 20 min at 37 °C, and added to a reaction mix containing 7.5 µL of nuclease-treated rabbit reticulocyte lysate (RRL, Promega, Madison, WI, USA), 1 mM amino acid mix minus methionine, and 6 µCi ³⁵S-methionine. Translation reactions were incubated at 30 °C for 90 min and treated with 1 µg of RNase A for 10 min at room temperature. Labelled proteins were visualized using a PhosphorImager screen (GE Healthcare, Chicago, IL, USA).

Fajardo T J.r., Sung P.Y., Celma C.C, & Roy P. (2017). Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity. Viruses, 9(7), 167.

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Radioactive DNA Oligonucleotide Binding Assay

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The assay was done as described in (22 (link)). A 5’-[³²P]-labeled single-stranded DNA oligonucleotide containing a 3’-phosphotyrosine (N14Y, 5′-GATCTAAAAGACTTY, Midland Certified Reagents Company) was incubated at 1 nM with whole cell extract for 15 min at room temperature in buffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 40 µg/mL BSA, and 0.01% Tween-20. Reactions were terminated by the addition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mM EDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue]. Samples were subjected to a 16% denaturing PAGE. Gels were dried and exposed to a PhosphorImager screen (GE Healthcare). Gel images were scanned using a Typhoon™ FLA 9500 (GE Healthcare).

Al Abo M., Sasanuma H., Liu X., Rajapakse V.N., Huang S.Y., Kiselev E., Takeda S., Plunkett W, & Pommier Y. (2017). TDP1 is critical for the repair of DNA breaks induced by sapacitabine, a nucleoside also targeting ATM- and BRCA-deficient tumors. Molecular cancer therapeutics, 16(11), 2543-2551.

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Northern Blot Analysis of RNA Expression

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5–10 μg of total RNA was separated on a 1.5% denaturing agarose-formaldehyde gel and transferred to a positively charged nylon membrane (Roche Applied Bioscience or PerkinElmer, Waltham, MA). Northern probes were generated with gene specific primers by amplifying ~ 200–500 bp of transcript from genomic DNA. Primer sequences are given in S4 Table. 100–150 ng of each DNA probe was labeled using the High Prime DNA Labeling Kit (Roche Applied Bioscience) and [α – ³²P] dCTP (PerkinElmer). Membranes were blocked in UltraHybe hybridization buffer (Ambion, Life Technologies) for at least 30 minutes and denatured probe was added to the same blocking buffer and incubated at 42°C overnight. The next day membranes were washed twice in 2X SSC, 0.1% SDS for 5 minutes and twice in 0.1X SSC, 0.1% SDS for 15 minutes at 42°C before exposure to a phosphorimager screen (GE Life Sciences, Pittsburgh, PA). Screens were scanned with a Fuji FLA-5100 imager and analyzed with Multi Gauge Software (ver. 3.1; Fujifilm/GE Life Sciences).

Gilmore S.A., Voorhies M., Gebhart D, & Sil A. (2015). Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma. PLoS Genetics, 11(7), e1005395.

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Quantifying Protein-RNA Interactions

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Five micrograms of protein lysate was incubated with 1.4 pmol of radiolabeled probe in a final volume of 20 μl. Reactions were incubated at 22°C for 20 min in 1× reaction buffer consisting of 100 mM KCl, 10 mM Tris pH 7.6, 5 mM MgCl₂, 1 mM DTT, 500 ng Poly (I)-(C) (ThermoFisher Waltham, MA, USA). Reactions with unlabeled specific wild-type (wt) competitor and non-specific tRNA competitor at 10, 25, 50 and 100× molar excess were carried out in parallel. The 20 μl reaction was loaded onto a 1× Tris Borate EDTA buffer, 6% non-denaturing polyacrylamide gel and electrophoresed for 2.5 h. Gels were transferred to Whatman 3CHR paper (Whatman, Maidstone UK), dried at 80°C under vacuum for 2 h, and exposed to a phosphorimager screen (GE Healthcare Life Sciences, Little Chalfont, UK). At least three independent assays were performed per probe. Typhoon 9410 software was used to analyze the gels and determine relative signal intensities of the shifted bands. Background subtracted signal intensity for each sample was determined as a ratio to wt in the absence of competitor. Relative average intensities were calculated and compared by paired t-tests using GraphPad Prism.

Sztuba-Solinska J., Diaz L., Kumar M.R., Kolb G., Wiley M.R., Jozwick L., Kuhn J.H., Palacios G., Radoshitzky S.R., J. Le Grice S.F, & Johnson R.F. (2016). A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8. Nucleic Acids Research, 44(20), 9831-9846.

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Analysis of TDP1 Enzyme Activity

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A 50′-[³²P]-labeled single-stranded DNA oligonucleotide containing a 3′-phosphotyrosine (N14Y)^{28 (link)} was incubated at 1 nM with 10 pM recombinant TDP1 in the absence or presence of inhibitor for 15 min at room temperature in the LMP1 assay buffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01% Tween-20.^{32 (link)} Reactions were terminated by the addition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mM EDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue]. Samples were subjected to a 16% denaturing PAGE with multiple loadings at 12 min intervals. Gels were dried and exposed to a PhosphorImager screen (GE Healthcare). Gel images were scanned using a Typhoon 8600 (GE Healthcare), and densitometry analyses were performed using ImageQuant software (GE Healthcare).

Beck D.E., Abdelmalak M., Lv W., Reddy P.V., Tender G.S., O’Neill E., Agama K., Marchand C., Pommier Y, & Cushman M. (2015). Discovery of Potent Indenoisoquinoline Topoisomerase I Poisons Lacking the 3-Nitro Toxicophore. Journal of medicinal chemistry, 58(9), 3997-4015.

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Tdp1 Activity Assay with Inhibitor

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A 5′-[³²P]-labeled single-stranded DNA oligonucleotide containing a 3′-phosphotyrosine (N14Y) was incubated at 1 nM with 10 pM recombinant Tdp1 in the absence or presence of inhibitor for 15 min at room temperature in the LMP1 assay buffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01% Tween-20. Reactions were terminated by the addition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mM EDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue]. Samples were subjected to a 16% denaturing PAGE with multiple loadings at 12 min intervals. Gels were dried and exposed to a PhosphorImager screen (GE Healthcare). Gel images were scanned using a Typhoon 8600 (GE Healthcare), and densitometry analyses were performed using the ImageQuant software (GE Healthcare).

Wang P., Elsayed M.S., Plescia C.B., Ravji A., Redon C.E., Kiselev E., Marchand C., Zeleznik O., Agama K., Pommier Y, & Cushman M. (2017). Synthesis and Biological Evaluation of the First Triple Inhibitors of Human Topoisomerase 1, Tyrosyl–DNA Phosphodiesterase 1 (Tdp1), and Tyrosyl–DNA Phosphodiesterase 2 (Tdp2). Journal of medicinal chemistry, 60(8), 3275-3288.

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TDP1 Inhibitor Assay Protocol

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A 5′-[³²P]-labeled single-stranded DNA oligonucleotide containing a 3′-phosphotyrosine (N14Y)^{61 (link)} was incubated at 1 nM with 10 pM recombinant TDP1 in the absence or presence of inhibitor for 15 min at room temperature in the LMP1 assay buffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01% Tween-20.^{62 (link)} Reactions were terminated by the addition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mM EDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue]. Samples were subjected to a 16% denaturing PAGE with multiple loadings at 12 min intervals. Gels were dried and exposed to a PhosphorImager screen (GE Healthcare). Gel images were scanned using a Typhoon 8600 (GE Healthcare), and densitometry analyses were performed using the ImageQuant software (GE Healthcare).

Elsayed M.S., Su Y., Wang P., Sethi T., Agama K., Ravji A., Redon C.E., Kiselev E., Horzmann K.A., Freeman J.L., Pommier Y, & Cushman M. (2017). Design and Synthesis of Chlorinated and Fluorinated 7-Azaindenoisoquinolines as Potent Cytotoxic Anticancer Agents That Inhibit Topoisomerase I. Journal of medicinal chemistry, 60(13), 5364-5376.

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Murine Apmap mRNA Expression Analysis

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Total RNA (10 µg) was resolved by formaldehyde/agarose gel electrophoresis and
blotted onto a Hybond-N⁺ membrane (GE Healthcare, Waukesha, WI, USA). The
mRNA was hybridized with a murine Apmap cDNA probe generated by PCR (primer located
in the middle 5′-CCAGGATCCTGTTGGACCAGTTGCAGTTC-3′ and at the 3′
end of the Apmap coding sequence) and labeled with [α-[³²P]]dATP
(GE Healthcare) using the Prime-a-Gene DNA labeling kit according to the
manufacturer’s protocol (Promega, Madison, WI, USA). After hybridization and
washing, signals were visualized by exposure to a PhosphorImager Screen (GE
Healthcare). Transcripts discussed in the results section were obtained from GenBank
[National Center for Biotechnology Information (NCBI), Bethesda, MD, USA; https://www.ncbi.nlm.nih.gov/genbank/) and The
European Bioinformatics Institute (Ensembl), Hinxton, United Kingdom; http://www.ensembl.org.].

Pessentheiner A.R., Huber K., Pelzmann H.J., Prokesch A., Radner F.P., Wolinski H., Lindroos-Christensen J., Hoefler G., Rülicke T., Birner-Gruenberger R., Bilban M, & Bogner-Strauss J.G. (2017). APMAP interacts with lysyl oxidase–like proteins, and disruption of Apmap leads to beneficial visceral adipose tissue expansion. The FASEB Journal, 31(9), 4088-4103.

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CFGE Analysis of Telomeric DNA

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CFGE was performed as described previously [14] . Cells were imbedded in 0.7% agarose, lysed with 0.5% SDS in Tris-HCl, and digested with RNase A (100 μg/ml) and proteinase K (250 μg/ml) at 37°C overnight. Gel electrophoresis was performed using 0.7% agarose in TAE buffer. In-gel hybridization analysis of telomeric DNA was performed as follows: the gel was dried for 1 hour at room temperature and hybridized overnight at 42°C with a telomeric probe in 1× hybridization buffer (2 μg/ml sonicated Escherichia coli DNA, 10× Denhards’ buffer, 0.5% SDS, and 5× SSC). The gel was washed four times in wash buffer (2× SSC, 0.5% SDS) and exposed to a PhosphorImager screen (GE Healthcare Life Science).

Liu H., Xie Y., Zhang Z., Mao P., Liu J., Ma W, & Zhao Y. (2018). Telomeric Recombination Induced by DNA Damage Results in Telomere Extension and Length Heterogeneity. Neoplasia (New York, N.Y.), 20(9), 905-916.

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