Fla 5000 phosphoimager

Manufactured by Fujifilm

Sourced in Japan

The FLA-5000 phosphoimager is a scientific instrument used for the detection and analysis of radioactive samples. It is designed to capture high-resolution images of phosphor-labeled biomolecules, such as proteins and nucleic acids, allowing researchers to quantify and study their distribution and abundance. The FLA-5000 utilizes a sensitive laser scanning technology to provide accurate and reproducible results, making it a valuable tool for a wide range of applications in molecular biology, biochemistry, and biomedical research.

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Lab products found in correlation

Yeast trna, by Thermo Fisher Scientific (2 mentions) T4 pnk, by New England Biolabs (2 mentions) Gel dryer, by Bio-Rad (2 mentions) 35s met cys pro mix, by PerkinElmer (1 mentions) Image studio software, by LI COR (1 mentions) Rnasin, by Thermo Fisher Scientific (1 mentions) Imaging plate, by Fujifilm (1 mentions) T4 polynucleotide kinase, by GE Healthcare (1 mentions) Hybond n, by GE Healthcare (1 mentions) Illustra microspin g 25 column, by GE Healthcare (1 mentions) γ 32p atp, by Hartmann Analytic (1 mentions) Tnt t7 coupled wheat germ extract system, by Promega (1 mentions) Complete edta free protease inhibitor, by Roche (1 mentions) Sybr gold, by Thermo Fisher Scientific (1 mentions) Ultrahyb oligo, by Thermo Fisher Scientific (1 mentions) Novex tbe urea gel, by Thermo Fisher Scientific (1 mentions) T4 polynucleotide kinase, by New England Biolabs (1 mentions) γ 32p atp, by PerkinElmer (1 mentions)

11 protocols using fla 5000 phosphoimager

Assessing Compound Effects on Cell Viability and Protein Synthesis

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The AlamarBlue (AB) reagent (Life Technologies, Carlsbad, CA) was used to assess the effect of test compounds on cell viability. Seeded A549 cells were treated with compounds using a 10-point twofold serial dilution (50 to 0.1 µM) and incubated for 48 h. AB was added to a final concentration of 10% (v/v) and incubated for 4 h and fluorescence measured at an excitation/emission of 545/590 nm. The percentage reduction in AB was calculated using the following controls: 0% reduced (DMEM + AB) and 100% reduced (Cells + DMEM + AB).
To assess compound effect on global cellular protein synthesis, cells were treated with compound for 24 and 48 h prior to labeling with [³⁵S]Met/Cys pro-mix (PerkinElmer) for 1 h. To determine the effect of compound on SeV replication, cells were treated for 2 h followed by infection for 18 h prior to labeling as above. Whole-cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by isotope incorporation visualization and quantification using a FLA-5000 phosphoimager (FujiFilm, Tokyo, Japan) and Image Studio software (Li-Cor, Lincoln, NE).

Gage Z.O., Vasou A., Gray D.W., Randall R.E, & Adamson C.S. (2016). Identification of Novel Inhibitors of the Type I Interferon Induction Pathway Using Cell-Based High-Throughput Screening. Journal of Biomolecular Screening, 21(9), 978-988.

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UV Cross-Linking and RNA-Binding Assay

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Trichomonad cytoplasmic extracts and the recombinant proteins TvACTN3r, the TvACTN3 domains (DIr, DIIr, and DIIIr), hIRP-1r (used as a positive control), and BSA (1 μg) (used as a negative control) were incubated with 200,000 cpm (10–15 ng) ³²P-labeled RNA probes for 30 min at 4°C in 25 μL reaction buffer (10 mM HEPES-KOH [pH 7.4], 3 mM MgCl₂, 5% (v/v) glycerol, 100 mM KCl, 20 U RNasin, and 5 μg yeast tRNA; Invitrogen). After RNA-binding, the reaction mixture was placed on ice and irradiated with a UV-lamp (UVP; 800,000 μJ/cm²) for 15 min. Unprotected RNA was digested for 30 min at 25°C with RNase A (10 μg) and RNase T1 (20 U). RPCs were resolved on 10% SDS-PAGE gels. The gels were stained with Coomassie Brilliant Blue (CBB) and dried, and radioactive bands were visualized by autoradiography in a FLA 5000 phosphoimager (Fujifilm, Co, Tokyo Japan) with a Software MultiGauge V3.0 [22 (link)]. These experiments were independently performed at least three times, with similar results.

Calla-Choque J.S., Figueroa-Angulo E.E., Ávila-González L, & Arroyo R. (2014). α-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism. BioMed Research International, 2014, 424767.

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Probing hSSB1 Binding to DNA Structures

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The interaction of recombinant hSSB1 with G-rich strand (TTAGGG)₅, C-rich strand (AATCCC)₅ and GC-duplex oligonucleotides was investigated using native acrylamide electrophoretic mobility shift analysis. Increasing concentrations of hSSB1 were incubated with ³²P-labelled oligonucleotide ssDNA (50 pmol) in buffer (20mM HEPES, pH 7.3, 100mM KCl and 1mM MgCl2, 1 mg µl⁻¹ bovine serum albumin) at 20⁰ C for 30 min in 10 µL total volume. Reactions were resolved on 10% native acrylamide/TBE gel described previously (17 (link)). Gels were exposed to a phosphorimage plate and visualized with a Fuji FLA-5000 Phosphoimager.

Pandita R.K., Chow T.T., Udayakumar D., Bain A.L., Cubeddu L., Hunt C.R., Shi W., Horikoshi N., Zhao Y., Wright W.E., Khanna K.K., Shay J.W, & Pandita T.K. (2015). Single-strand DNA binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs. Cancer research, 75(5), 858-869.

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Auxin Transport in Cut Flowers

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For determining the acropetal transport of NAA or 2,4-D by autoradiography, cut flowers were pulsed for 24 h with the standard auxin pulsing solutions containing 16.0 KBq of either [1-¹⁴C]NAA or [1-¹⁴C]2,4-D as tracers. At the end of a 24-h pulsing treatment (0 h), the lower part of each cut flower shoot was thoroughly washed with distilled water, and the cut flowers were dried off by pressing them for 1 week between 20 × 40-cm blotting papers at room temperature. The dried samples were exposed to Fujifilm Imaging Plates for 48 h, the images were then analyzed by a Fujifilm Fla-5000 phospho-imager, and processed with Photoshop version 7.0.

Abebie B., Philosoph-Hadas S., Riov J., Huberman M., Goren R, & Meir S. (2020). Raising the pH of the Pulsing Solution Improved the Acropetal Transport of NAA and 2,4-D and Their Efficacy in Reducing Floret Bud Abscission of Red Cestrum Cut Flowers. Frontiers in Plant Science, 11, 825.

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Substrate tRNA Binding Assay for TrcP

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The substrate tRNA binding capacities of TrcP and the KRmutant were evaluated using a gel mobility shift assay. Isolated tRNA-Tyr from the ΔtrcP strain and tRNA-Asp from the WT strain were labeled with ³²P at their respective 5′ ends. Briefly, calf intestinal alkaline phosphatase (CIP) (NEB) was used to dephosphorylate tRNAs, which were purified with gel electrophoresis, and labeled with [γ-³²P]ATP by T4 PNK (NEB), followed by gel purification. WT or KRmut TrcP proteins (0–5 μM) were incubated with 1 μM cold tRNA-Tyr or tRNA-Asp and a trace amount of ³²P-labeled tRNAs (<0.1 μM) in a 5 μl reaction in 5 mM MgCl₂, 50 mM Tris-HCl pH 8.0, and 60 mM NaCl at 37 °C for 10 min, followed by mixing with loading dye (80% glycerol and 0.05% bromophenol blue) and gel electrophoresis for 30 min at 180 V and 4 °C on 4% polyacrylamide gel containing 50 mM Tris-Acetic acid pH 8.0 and 5 mM Mg(Oac)₂; the running buffer was the same composition as the gel. Gels were dried with a gel dryer (Biorad) and bands were detected using an FLA-5000 phosphoimager (Fuji Film).

Kimura S., Srisuknimit V., McCarty K.L., Dedon P.C., Kranzusch P.J, & Waldor M.K. (2022). Sequential action of a tRNA base editor in conversion of cytidine to pseudouridine. Nature Communications, 13, 5994.

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RNA-Protein Complex Formation Assay

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Ten pmol in vitro transcribed RNA were 5’ labeled with γ-³²P-ATP. Per lane, 0.5 μM unlabelled scr5239 RNA mixed with 50 kcpm of the ³²P-labeled scr5239 RNA were incubated with 7 or 14 μM unlabeled M1 RNA in a 10 μl reaction containing 10 mM Tris pH 7.0, 100 mM KCl, 10 mM MgCl₂ and 1 μg yeast tRNA (Invitrogen) to prevent unspecific binding. After 15 min incubation at 28°C each sample was mixed with 5 μl 5x native loading buffer (50% glycerol, 0.2% bromophenol blue, 0.5x TB) and loaded onto a running 6% native PAA gel—precooled to 4°C—with 0.5x TB as running buffer. After the run, the gel was dried for 30 min at 80°C. Radioactivity was detected using a FUJI FLA-5000 Phosphoimager. For competition assays 10 μM of the oligonucleotide 1-E (S1 Table) were added prior to the incubation.

Vockenhuber M.P., Heueis N, & Suess B. (2015). Identification of metE as a Second Target of the sRNA scr5239 in Streptomyces coelicolor. PLoS ONE, 10(3), e0120147.

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Quantification of scr5239 RNA Expression

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A total of 30 μg total RNA was separated on 6% denaturing polyacrylamide gels and transferred to a positively charged nylon membrane (Hybond-N+, GE Healthcare) in a tank blotting device (Peqlab) at 4°C. As probe for detection, 10 pmol oligonucleotide were radiolabeled at the 5′ end using 5 μl γ-[³²P] ATP (∼3.3 pmol/μl, Hartmann-Analytic) and 1 μl T4 polynucleotide kinase (Roche) in the supplied buffer for 1 h at 37°C and subsequently purified using Illustra MicroSpin G-25 columns (GE Healthcare). 25 μl radiolabeled oligonucleotides (approximately 300 kc.p.m./μl) where used as probe for each experiment. Signals were quantified by phosphoimaging using a FLA-5000 phosphoimager (Fujifilm life Science). Expression of scr5239 (probe: scr5239_A) was normalized to the amount of 5S rRNA (probe: 5S_A).

Engel F., Ossipova E., Jakobsson P.J., Vockenhuber M.P, & Suess B. (2020). sRNA scr5239 Involved in Feedback Loop Regulation of Streptomyces coelicolor Central Metabolism. Frontiers in Microbiology, 10, 3121.

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Affinity Purification of Labeled Proteins

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Recombinant SE and GFP proteins fused to maltose-binding protein (MBP) were purified from Escherichia coli as described before (11 ). [³⁵S]-methionine labeled HEN2, ZCCHC8A, ZCCHC8B and RBM7 proteins were obtained by in vitro transcription and translation using the TNT T7 coupled wheat germ extract system (Promega) following the protocol supplied by the manufacturer. Purified MBP-SE and MBP-GFP were bound to amylose beads (NEB) by overnight incubation at 4°C. Excess protein was removed by washing the beads three times with 20 mM Tris HCl pH 7.5, 200 mM NaCl, 1 mM EDTA, cOmplete™ EDTA-free protease inhibitor (Roche) before the radiolabeled proteins were added. After incubation for 2 h at 4°C the beads were washed five times and proteins were eluted with 20 mM maltose. Eluted proteins were separated by 12% SDS-PAGE and detected using a FLA-5000 phosphoimager (Fujifilm).

Bajczyk M., Lange H., Bielewicz D., Szewc L., Bhat S.S., Dolata J., Kuhn L., Szweykowska-Kulinska Z., Gagliardi D, & Jarmolowski A. (2020). SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis. Nucleic Acids Research, 48(12), 6839-6854.

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Evaluation of tRNA Binding Affinity

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The substrate tRNA binding capacities of TrcP and the KRmutant were evaluated using a gel mobility shift assay. Isolated tRNA-Tyr from the ΔtrcP strain and tRNA-Asp from the WT strain were labeled with 32 P at their respective 5' ends. Briefly, calf intestinal alkaline phosphatase (CIP) (NEB) was used to dephosphorylate tRNAs, which were purified with gel electrophoresis, and labeled with [γ-32 P]ATP by T4 PNK (NEB), followed by gel purification. WT or KRmut TrcP proteins (0-5 μM) were incubated with 1 μM cold tRNA-Tyr or tRNA-Asp and a trace amount of 32 P-labeled tRNAs ( < 0.1 μM) in a 5 μl reaction in 5 mM MgCl2, 50 mM Tris-HCl pH 8.0, and 60 mM NaCl at 37 °C for 10 min, followed by mixing with loading dye (80% glycerol and 0.05% bromophenol blue) and gel electrophoresis for 30 min at 180 V and 4 °C on 4% polyacrylamide gel containing 50 mM Tris-Acetic acid pH 8.0 and 5 mM Mg(OAc)2; the running buffer was the same composition as the gel. Gels were dried with a gel dryer (Biorad) and bands were detected using an FLA-5000 phosphoimager (Fuji Film).

Kimura S., Srisuknimit V., McCarty K., Dedon P.C., Kranzusch P.J., & Waldor M.K. (2022). Sequential action of a tRNA base editor in conversion of cytidine to pseudouridine.

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Autoradiography of Radiolabeled Transcripts

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Gels with radioactive samples were exposed to an Imaging Plate for autoradiography. Signals derived from radiolabeled RNA transcripts or labeled DNA were detected with FUJIFILM FLA 5000 PhosphoImager (Fuji, Japan) and analysed with Aida Image software v4.27 (Raytest, Straubenhardt, Germany).

Dexl S., Reichelt R., Kraatz K., Schulz S., Grohmann D., Bartlett M, & Thomm M. (2018). Displacement of the transcription factor B reader domain during transcription initiation. Nucleic Acids Research, 46(19), 10066-10081.

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