The DNA labeling reaction mixture contained a buffer (50 mM Tris–HCl pH 7.6, 10 mM MgCl2, 5 mM of DTT, 0.1 mM spermidine), 10 U T4 polynucleotide kinase, 20 mCi of (γ-32P)ATP with an activity of 5000 Ci/mmol (Hartmann Analytic, Braunschweig, Germany) and 20 pmol of a DNA oligomer (Table S1 ). After an hour-long incubation at 37 °C, the 32P labelled products were purified using a G25 column (Sigma-Aldrich, St. Louis, MO, USA). The radioactivity level of the labeled molecules was measured using a scintillation counter. The total RNA isolated from the different cultures was separated on 12% (w/v) polyacrylamide gels, electrotransferred onto Hybond-Nylon membranes (GE Healthcare, Chicago, IL, USA), crosslinked with UV light (120 mJ/cm2) and prehybridized in a 2xSSC, 1X Denhard solution for 1 h at 37 °C. Hybridization of 32P labelled DNA probes (20 × 106 cpm) was carried out at 42 °C overnight in PerfectHybTM Plus solution (Sigma). Then, the hybridization mixture was discarded, and the blot was washed in the same solution several times, until the radioactivity in the solution disappeared. Finally, the blots were analyzed using phosphor imaging screens and a FLA-5100 image analyzer with MultiGauge software (FujiFilm).
Fla 5100 image analyzer
Manufactured by Fujifilm
Sourced in Japan
The FLA-5100 image analyzer is a specialized lab equipment designed for the analysis and quantification of images. It is capable of capturing and processing images from a variety of sources, including gels, blots, and other sample types. The FLA-5100 provides precise measurements and data analysis to support research and scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
γ 32p atp, by Hartmann Analytic (1 mentions)
Hybond nylon membrane, by GE Healthcare (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Hybond, by Cytiva (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Lys c, by Thermo Fisher Scientific (1 mentions)
Originpro software, by OriginLab (1 mentions)
Multi gauge, by Fujifilm (1 mentions)
Rabbit reticulocyte lysate system, by Promega (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
6 protocols using fla 5100 image analyzer
1
Radioactive DNA Labeling and Northern Blot
2
Kinase Activity Profiling by Mass Spectrometry
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Purified recombinant proteins were incubated with [g-32 P] ATP for 30 min at room temperature in the kinase assay buffer (50 mM HEPES [pH 7.4], 1 mM DTT, 10 mM MgCl 2 , 0.6 mM unlabeled ATP). The mixture was subsequently separated by SDS-PAGE, and autoradiography was detected by FLA-5100 image analyzer (Fujifilm, Japan). For identification of in vitro phosphorylation sites by LC-electrospray ionization (ESI)-MS/MS, 1.5 mM unlabeled ATP was used in the kinase buffer. The proteins were separated by SDS-PAGE, followed by Coomassie Brilliant Blue staining and were digested by trypsin (Thermo Fisher Scientific, catalog no. 90057) or Lys-C (Thermo Fisher Scientific, catalog no. 90051).
3
In Vitro Kinase Assay Protocol
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For in vitro kinase assay, the KIN3 cytoplasmic domain (178 to 510 aa) was amplified from cDNA and inserted into the expression vector pGEX-6p-1 between the BamHI and XhoI restriction sites. DNA fragment corresponding to the AMK24 cytoplasmic domain (49 to 432 aa) was amplified and cloned into pMAL-c2X between the XbaI and SalI restriction sites. Various mutations (KIN3KDK322R, KIN3KDD415N, KIN3KDD433N, and AMK24KDK143R) were introduced by overlap extension PCR. The constructs were transferred into E. coli strain BL21 to purify the recombinant proteins. About 0.5 µg of GST-fused recombinant proteins alone or in combination with 0.5µg of MBP-fused recombinant proteins were incubated in the 20 µL reaction mixture [50 mM Tris-HCl (pH8.0), 50 mM KCl, 5 mM MgCl2, 5 mM MnCl2, 1 mM DTT, and 5 μCi [γ-32P] ATP (10 μCi/µL)] at room temperature for 30 min. Casein (1 mg mL−1) served as a substrate in the kinase assay. The reactions were terminated by adding 5xSDS loading buffer and boiled for 10 min. Samples were separated on a 10% SDS-PAGE and the gel was exposed to FLA-5100 image analyzer (Fuji Film). Coomassie brilliant blue-stained PAGE gels were used as loading control.
For autophosphorylation assays, the commercial anti-pSer/Thr (PP2551; ECM Biosciences; dilution 1:1,000) antibody was used for immunoblotting.
For autophosphorylation assays, the commercial anti-pSer/Thr (PP2551; ECM Biosciences; dilution 1:1,000) antibody was used for immunoblotting.
4
Characterizing 5'CS-Protein Interactions
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5′CS (fluorescently labeled with Cyanine 3), 3′CS and 5′CS partner RNA oligonucleotides were separately denatured for 5 min at 95°C and chilled on ice. Subsequently, 2 pmol of 5′CS and 5 pmol of 5′CS partner were mixed in 1× folding buffer and incubated for 10 min at 65°C and 5 min on ice to form the initial 5′CS duplex. Next, 5 pmol of 3′CS oligomer and increasing concentrations of protein were added and samples were incubated for 10 min at 37°C. Reaction was terminated by addition of SDS (0.25% final concentration). Samples were analyzed on native 12% gel (19:1 acrylamide/bisacrylamide ratio) in 0.5× TB at 4°C (DNAPointer, BioVectis). Gels were scanned with FLA5100 image analyzer (FujiFilm) and data from at least three independent experiments were quantified using MultiGauge (FujiFilm) and OriginPro software (Origin Lab).
5
Monitoring RNA-RNA Complex Formation
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For monitoring of RNA–RNA complexes’ formation, 5 pmol of H82-135 (or its mutants) and 50 pmol of 3′CS oligo or 5 pmol of D1627–1872 and 50 pmol of H56–169 (or its mutants) RNA were mixed together, renatured for 5 min at 75°C, and slowly cooled (0.1°C/sec) to 4°C. Next, samples were incubated in 1× folding buffer for 20 min at room temperature, placed on ice, and glycerol was added to the final concentration of 1%. Samples were analyzed by native gel electrophoresis using 12% gel (19:1 acrylamide/bisacrylamide ratio) in 0.5× TB at 4°C (DNAPointer, BioVectis). RNA was visualized using SYBR Gold staining and scanned with FLA5100 image analyzer (FujiFilm).
6
In Vitro Protein Translation Assay
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In vitro translation was performed using Rabbit Reticulocyte Lysate (RRL) System (Promega). The reaction contained 8.75 μl RRL, 20 μM aminoacid mixture minus methionine, 0.5 μl 35S-methionine (1000 Ci/mol, Hartman Analytic), 10 U of RiboLockRNase inhibitor (Thermo Fisher Scientific) and 1.25 pmol of capped RNA, which was previously denatured for 5 min at 65°C. The final volume of the reaction was 12.5 μl and it was conducted for 90 min at 30°C. Afterwards, the reaction was treated with 0.16 μg of RNase A for 5 min at 20°C, and denatured for 2 min at 80°C in the presence of SDS Sample Buffer and 100 mM DTT. The reaction products were analyzed in 15% SDS-PAGE, followed by radioisotope imaging with FLA 5100 image analyzer (FujiFilm).
For translation inhibition assay, RRL was pre-incubated with an increasing concentration of m7GpppG cap analog from 5 to 750 μM (Epicenter Biotechnologies) and the same concentrations of magnesium acetate, for 15 min at 30°C. Afterwards, 20 μM amino acid mixture minus methionine, 0.5 μl 35S-methionine (1000 Ci/mol, Hartman Analytic), 10 U of RiboLockRNase inhibitor (Thermo Fisher Scientific) and 1.25 pmol of previously denatured RNA were added to the RRL samples and incubated for 90 min at 30°C. The samples were treated with RNase A and analyzed in 15% SDS-PAGE as described above.
For translation inhibition assay, RRL was pre-incubated with an increasing concentration of m7GpppG cap analog from 5 to 750 μM (Epicenter Biotechnologies) and the same concentrations of magnesium acetate, for 15 min at 30°C. Afterwards, 20 μM amino acid mixture minus methionine, 0.5 μl 35S-methionine (1000 Ci/mol, Hartman Analytic), 10 U of RiboLockRNase inhibitor (Thermo Fisher Scientific) and 1.25 pmol of previously denatured RNA were added to the RRL samples and incubated for 90 min at 30°C. The samples were treated with RNase A and analyzed in 15% SDS-PAGE as described above.
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