Pelleted THP-1 cells were lysed for 30 min at 4 °C in lysis buffer (25 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM DTT, 2 mM EDTA, 0.5 mM PMSF, 0.05% NP-40, RNase inhibitor). After sonication, the lysates were centrifuged for 30 min at 13,000 × g at 4 °C. anti-SAMHD1 and anti-rabbit IgG antibodies were incubated with Dynabeads for 1 h at room temperature. The conjugated beads were washed sequentially twice each with buffer A (500 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.05% NP-40, RNase inhibitor) and buffer B (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.05% NP-40, RNase inhibitor). Cell lysates were incubated with the prepared conjugated Dynabeads for 1 h at 4 °C. The immunoprecipitates were washed five times with buffer B, followed by an in vitro nuclease assay. The in vitro nuclease assay was performed as described previously15 (link). In brief, immunoprecipitated proteins were incubated in 20 µl reaction mixtures containing phosphate-buffered saline (PBS) supplemented with 5 mM MgCl2, 2 mM dithiothreitol (DTT) and [γ−32P]-labeled RNA substrate (A20) at 37 °C for 30 min. The reactions were stopped by the addition of 20 µl of 2 × RNA loading buffer and then boiled at 95 °C for 5 min. RNA was separated on 8 M urea/15% polyacrylamide gels and analyzed using a BAS-2500 phosphorimager (Fujifilm).
Bas2500 phosphorimager
Manufactured by Fujifilm
Sourced in Japan
The BAS2500 is a phosphorimager manufactured by Fujifilm. It is a device used for the detection and quantification of radioactive signals in biological samples, such as those used in autoradiography and phosphor imaging experiments.
Automatically generated - may contain errors
Lab products found in correlation
α 32p dctp, by PerkinElmer (3 mentions)
Certified megabase agarose gel, by Bio-Rad (2 mentions)
Random primer dna labeling kit ver 2, by Takara Bio (2 mentions)
Chef dr 2, by Bio-Rad (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Image gauge 4, by Fujifilm (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Fastgene gel pcr extraction kit, by Nippon Genetics (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Match marker gold yeast two hybrid system, by Takara Bio (1 mentions)
Gel dryer, by Bio-Rad (1 mentions)
Gs gene linker, by Bio-Rad (1 mentions)
A7906, by Merck Group (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Nytran nylon membranes, by Cytiva (1 mentions)
Image gauge software, by Fujifilm (1 mentions)
35s methionine and cysteine, by PerkinElmer (1 mentions)
17 protocols using bas2500 phosphorimager
1
SAMHD1 RNase Activity Assay
2
Serum Stability Assessment of siRNAs
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Serum stability of siRNAs was assessed by northern blot analysis. siRNA (2 μM) was incubated in 45% human plasma at 37°C for indicated times prior to northern blot analysis by a conventional method. Briefly, total RNA extracted with Trizol LS reagent (Invitrogen) was separated by 15% polyacrylamide/7 M urea gel electrophoresis and transferred to a membrane by electroblotting. As a probe, 5′-end 32P-labeled, synthetic ribo-oligonucleotide complementary to siRNA guide-strand was used. Densitometric quantification of the radioactive hybridization signal was performed using a Fuji BAS-2500 Phosphorimager.
3
Measuring NUP153-RNA Binding Inhibition
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The DNA template used to synthesize the RNA probe was prepared by hybridizing the following DNA oligonucleotides: 5ʹ-TAATACGACTCACTATAG-3ʹ and 5ʹ-GGCGGCCGCTCTTGAACTAGTGGATCCCCCGGGCTGCACTATAGTGAGTCGTATTA-3ʹ. The resulting template was used for in vitro transcription with the T7MEGAscript Kit (Life Technologies; Grand Island, NY, USA) and [α-32P]UTP to produce an internally labeled version of B38, a 38-nucleotide RNA that is derived from pBluescript-SK(+) and has been shown to bind to the NUP153-RBD 17 (link). Gel-purified radioisotope-labeled B38 (20 fmol) was incubated for 1 hour at 4 °C with the NUP153-RBD (2 μM), which had been purified by Ni-affinity chromatography as described previously 17 (link), in a total volume of 10 μL of binding buffer containing 25 mM HEPES (pH 7.5), 100 mM KCl, 0.5% Triton X-100, 0.5 mg/mL bovine albumin, and 7.5% glycerol. For the competitive electrophoretic mobility shift assay (EMSA), increasing amounts (0.04, 0.4, and 1.0 mM) of NUP153 inhibitors were preincubated with the NUP153-RBD for 1 hour at 4 °C prior to incubation with the labeled B38. Samples were electrophoresed at 4 °C on a pre-run 20% native acrylamide gel. Phosphorimage analysis and quantification was performed using a Fuji BAS-2500 phosphorimager. The experiment was conducted three times independently.
4
Guanylyltransferase Activity Assay
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gRNA-transfected Cas9 cells or Cas9-ama cells were harvested at 2 days after electroporation. Cell pellets were stored at -80°C until use. The cells were resuspended in a buffer containing 50 mM Tris-HCl (pH 7.5), 20 mM NaCl, 10% sucrose, 0.1% Triton X-100 and 1×cOmplete EDTA-free Protease Inhibitor Cocktail (F. Hoffmann-La Roche, Ltd., Switzherland) for lysis, and cell debris were cleared by a centrifugation for 1 min at 8000 rpm. Guanylyltransferase assay was carried out by incubating 4 μg of cleared lysate in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 2 mM DTT and 40 μM [α-32P]-GTP for 20 min at 30°C[28 (link)]. The reaction was terminated by addition of SDS loading buffer, and the products were resolved on a 10% SDS-PAGE. Radiolabeled enzyme-GMP covalent adducts were visualized by BAS-2500 phosphorimager and quantitated by Image Gauge 4.0 software (Fujifilm Corp., Japan).
5
Postmortem Brain Autoradiography of AD
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3H-BU99008 and 3H-Deprenyl autoradiography studies were performed on large frozen postmortem brain sections from one CN, one patient with sporadic AD and one AβPPArc carrier case. The large frozen sections were allowed to dry at room temperature (RT) for 30–45 mins, followed by 1 h incubation with either 3H-BU99008 (1 nM) or 3H-Deprenyl (10 nM) at RT. Afterwards, the sections were rinsed for 5 mins three times in cold buffer (50 mM Tris-HCl buffer, pH 7.4 for 3H-BU99008; 50 mM Na-K phosphate buffer, pH 7.4 for 3H-Deprenyl), followed by a quick dip in cold distilled water. The sections were allowed to dry at RT for 24 h and were then apposed together with a tritium standard (Larodan Fine Chemicals AB, Mälmo, Sweden) on a phosphor-plate for 4 and 7 days for 3H-Deprenyl and 3H-BU99008, respectively, and then imaged using a BAS-2500 phosphor imager (Fujifilm, Tokyo, Japan). For semiquantitative analyses, the regions of interest (ROI) were drawn manually using multigauge software on the autoradiogram and the photostimulated luminescence per square millimeter (PSL/mm2) was transformed into fmol/mg using the standard curve to determine the total, NSP and specific binding of 3H-BU99008 and 3H-Deprenyl in the ROI.
6
Quantitative Analysis of miRNA Tailing
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Terminal nucleotidyl transferase reactions were set up in a total volume of 25 μl containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 20 U of RNase inhibitor (Promega), 10 μCi of [α-32P] rNTP (3000 Ci/mmol, Amersham Pharmacia Biotech), 250 ng (33.7 pmol) of miRNA or 30 pmol ribonucleotide homopolymer (20-nt), and 3.75 pmol of purified GLD-2. After 30 min of incubation at 37°C, reactions were stopped by adding 60 μl of an acid phenol emulsion [phenol:chloroform:10% SDS:0.5 M EDTA (1:1:0.2:0.4)] and 20 μg of glycogen. RNA products were precipitated with 2.5 volumes of cold 5 M ammonium acetate-isopropanol (1:5) and washed with 80% cold ethanol. After heat denaturation, the RNA samples were subjected to electrophoresis on an 8 M urea–15% polyacrylamide gel and were visualized by autoradiography. Densitometric quantification of radioactivity was performed using a Fuji BAS-2500 PhosphorImager.
7
Quantitative Mature miRNA Measurement
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Splinted ligation assay was performed as described previously using total RNA (2 μg) extracted from each sample to measure mature miRNA levels [41 (link)-43 (link)]. Equal amounts of input RNA for reactions were further verified by visualizing 5.8S RNA as an internal control with ethidium bromide after electrophoresis of total RNA on denaturing polyacrylamide gels. The sequences of miRNA-specific bridge oligonucleotides used for the splinted ligation assay are listed in Additional file 2 . Reaction products were resolved on 12% polyacrylamide gels containing 7 M urea, visualized on a BAS-2500 Phosphorimager (Fujifilm), and quantified using MultiGauge software (Fujifilm).
8
Determination of Spontaneous GCR Rates
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Rates of spontaneous GCR were determined by means of fluctuation tests as previously described (35 (link),36 (link)). Pulse field gel electrophoresis (PFGE) was carried out using CHEF-DRII (Bio-Rad) under the following conditions: switching time 1800–1000 s, 2 V/cm for 45 h and then 70 s for 3 h at 10°C in 1 × TAE buffer (40 mM Tris-acetate, 1 mM EDTA) and in a 0.55% agarose gel (Certified megabase agarose gel, Bio-Rad) for broad-range PFGE; and switching time 40–70 s, 4.5 V/cm for 24 h at 10°C in 0.5 × TBE buffer and in 0.6% agarose gel (Certified megabase agarose gel, Bio-Rad) for short-range PFGE. Southern hybridization was done using Random Primer DNA Labeling Kit Ver.2 (TaKaRa) and [α-32P] dCTP (Perkin-Elmer). DNA bands were detected with a BAS2500 phosphorimager (Fuji film). For PCR analysis, DNA was recovered from agarose gel using a FastGene Gel/PCR extraction kit (Nippon Genetics).
9
Dissecting DCL1 Microprocessor Complex Activity
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Yeast two-hybrid assays were performed using the Matchmarker Gold Yeast Two-Hybrid System (Clontech, Kusatsu, Japan), according to the manufacturer's instructions. The FHA2 CDS was cloned into the pGADT7 vector containing the GAL4 activation domain. The DCL1 domains (Helicase, DUF283, PAZ, and RBD) were cloned into the pGBKT7 vector containing the GAL4 DNA-binding domain. Combinations of the recombinant pGADT7 and pGBKT7 plasmids were transformed into the AH19 yeast strain, and the growth of the yeast cells was observed in the selective media.
In vitro pri-miRNA processing assay
The DCL1-associated microprocessor complex was immunoprecipitated using an a-Myc antibody from 10-day-old 35S:DCL1-6Myc seedlings. In vitro DCL1 enzyme assays were performed in a total volume of 30 ml of reaction buffer (20 mM Tris-HCl [pH 7.5], 50 mM KCl, 4 mM MgCl 2 , 1 mM DTT, 7.5 mM ATP, 1 mM GTP, and 1 ml RNase Inhibitor [Ambion]) containing 1 ml of radiolabeled pri-miRNA ($2000 cpm) and 15 ml of the DCL1associated magnetic beads. The reaction mixture was incubated at 37 C for 3 h. After extraction with phenol-chloroform and precipitation with ethanol, the processing products were separated using 5%-15% denaturing PAGE. The denaturing gel was dried in a gel dryer (Bio-Rad) at 65 C for 2 h. The processed RNA products were detected using a BAS-2500 phosphorimager (Fujifilm, Tokyo, Japan).
In vitro pri-miRNA processing assay
The DCL1-associated microprocessor complex was immunoprecipitated using an a-Myc antibody from 10-day-old 35S:DCL1-6Myc seedlings. In vitro DCL1 enzyme assays were performed in a total volume of 30 ml of reaction buffer (20 mM Tris-HCl [pH 7.5], 50 mM KCl, 4 mM MgCl 2 , 1 mM DTT, 7.5 mM ATP, 1 mM GTP, and 1 ml RNase Inhibitor [Ambion]) containing 1 ml of radiolabeled pri-miRNA ($2000 cpm) and 15 ml of the DCL1associated magnetic beads. The reaction mixture was incubated at 37 C for 3 h. After extraction with phenol-chloroform and precipitation with ethanol, the processing products were separated using 5%-15% denaturing PAGE. The denaturing gel was dried in a gel dryer (Bio-Rad) at 65 C for 2 h. The processed RNA products were detected using a BAS-2500 phosphorimager (Fujifilm, Tokyo, Japan).
10
Southern Blot Analysis of DNA Fragmentation
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After EtBr staining, agarose gel was irradiated with 300 mJ ultraviolet (UV) light using GS Gene Linker (Bio-Rad) for DNA fragmentation, and then soaked into 800 mL of alkaline buffer (1.2 M NaCl, 0.4 M NaOH) for 40 min with gentle shaking to denature DNA. DNA was transferred to ClearTrans nylon membrane 0.45 µm (Wako, 039-22673) by capillary action in 25 mM sodium phosphate buffer (pH 6.5) and covalently attached to the membrane by 150 mJ UV irradiation. A 0.6 kb EcoRI–EcoRI fragment prepared from pTN75529 (link), α-32P-dCTP (PerkinElmer Life Sciences, NEG013H), and Random primer labeling kit ver. 2 (Takara, 6045) were used to prepare radioactive probe A. Radioactive signals were detected using BAS2500 phosphorimager (Fuji Film).
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