Yeast cells were diluted to an A600 = 0.02 in 1 ml of YPD in the presence of 2 μ Ci/ml 32Pi and cultivated to stationary phase. Phospholipids were extracted from total cells and separated by TLC with developing solvent A (chloroform/ethanol/water/triethylamine, 30/35/7/35, v/v) or solvent B (chloroform/methanol/25% ammonia, 65/35/5, v/v) (Tamura et al., 2013 (link)). After 2 to 3 hours developing, the TLC plates were dried and sandwiched with storage phosphor screens using an exposure cassette (GE healthcare). The RI-labeled phospholipids were detected by radioimaing with Typhoon FLA-7000 image analyzer (GE Healthcare).
Typhoon fla 7000 image analyzer
Manufactured by GE Healthcare
Sourced in United States
The Typhoon FLA 7000 is a versatile image analyzer designed for a range of applications in life science research. It provides high-sensitivity detection and quantification of fluorescently labeled proteins, nucleic acids, and other biomolecules separated by gel electrophoresis or membrane-based techniques. The system employs multiple laser excitation sources and sensitive fluorescence detection to capture high-quality images of labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Zeta probe, by Bio-Rad (2 mentions)
Perfecthyb plus hybridization buffer, by Merck Group (2 mentions)
Bcabest labeling kit, by Takara Bio (2 mentions)
Exposure cassette, by GE Healthcare (1 mentions)
Yeast rna, by Thermo Fisher Scientific (1 mentions)
Multi gauge software v3, by Fujifilm (1 mentions)
Ix71 fluorescence microscopy system, by Olympus (1 mentions)
Fp 6500 spectrofluorometer, by Jasco (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
6 protocols using typhoon fla 7000 image analyzer
1
Quantification of Yeast Phospholipids
2
RNA-RdRp Complex Formation Assay
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The 32P-labeled 5′-end-labeled RNA (1 pmol) and the RdRp (200, 400, 600 pmol) were incubated in the reaction buffer (50-mM Tris-HCl pH 7.4, 1.7% glycerol, 5-mM β-mercaptoethanol, and 0.5 μg/μl of yeast RNA [Thermo Fisher Scientific]) at 30 °C for 10 min (total volume, 20 μl). A 4 μl of 6× gel-loading buffer was then added to the reaction mixture. The RNA and RdRp complex was separated with native PAGE at a constant voltage of 60 V for 90 min at 4 °C in the 1/2 × running buffer (12.5-mM Tris, 86-mM glycine, pH 8.3) after the gel was prerun at a constant voltage of 60 V for 30 min at 4 °C and analyzed by Typhoon FLA 7000 image analyzer (GE Healthcare). In the case of the competition assay, the procedures are the same as above except that the nonlabeled RNA (0, 2.4, 4.8, 12.0, or 24.0 pmol) was incubated with 32P-labeled 100AS RNA (0.2 pmol) and the RdRp (600 pmol), respectively.
3
RNA Northern Blot Analysis of siRNA
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Approximately 5 µg of total RNA isolated using the Trizol reagent was electrophoresed in 18% denaturing polyacrylamide gels containing 1×TBE buffer [89 mM Tris (pH 8.3), 89 mM boric acid, 2 mM EDTA] and 7 M urea, and blotted onto a nylon membrane (Zeta-Probe, Bio-Rad, USA) by electroblotting (1.0 mA/1.0 cm2 membrane for 1 h at 4°C). DNA fragments of the GFP gene and the CaMV 35S promoter as probes were amplified by PCR, and then probes for siRNA detection were made using the BcaBEST Labeling Kit (Takara Bio) with [α-32P]dCTP. PCR primers are listed in Supplementary Table S1. Hybridization was carried out in Perfect Hyb Plus hybridization buffer (Sigma-Aldrich) containing 32P-labeled DNA probe at 45°C for 6 h. Membranes were washed four times in 2×SSC (1×SSC, 0.15 M NaCl, 15 mM sodium citrate) with 0.5% SDS at 45°C for 1 h, and then analyzed by a Typhoon FLA 7000 image analyzer (GE Healthcare) (Fukuhara et al. 2011 (link)).
4
Hybridization and Analysis of ErbB2 Aptamer
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Hybridization was performed using the previously described protocol [31 (link)]. The sequence of the complementary oligonucleotide (cODN) platform was 3’-GTCGGTGTGGTGGTC-5’ and Cy-5 was labeled to the 5’-end of cODN. The reverse complement sequence (‘5-CAGCCACACCACCAG-3’ ) was attached to 3’-end of ErbB2 aptamer or scrambled aptamer for the base-pair hybridization with cODN. The hybridization of ErbB2 aptamer was performed in annealing buffer (10 mM Tris pH 7.5, 1 mM EDTA, 50 mM NaCl, 10 mM MgCl2). The mixture was incubated at 95 ºC for 5 min and hybridization efficiency of Cy5-hyErbB2 aptamer was analyzed by electrophoresis in 3% (w/v) agarose gel. Gels were imaged with a Typhoon FLA 7000 image analyzer (GE Healthcare, WI, USA) and analyzed using Multi Gauge software v3.0 (Fujifilm, Tokyo, Japan).
5
Northern Blot Analysis of Small RNAs
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Approximately 5 μg of total RNA was electrophoresed in 18% denaturing polyacrylamide gels containing 1× TBE buffer [89 mm Tris (pH 8.3), 89 mm boric acid, 2 mm EDTA] and 7 m urea, and blotted onto a nylon membrane (Zeta‐Probe, Bio‐Rad, Hercules, CA, USA) by electroblotting (1.0 mA/1.0 cm2 membrane for 1 h at 4°C). DNA fragments of PVCV and the CHS‐A gene as probes were amplified by PCR, and then probes for siRNA detection were made using the BcaBEST Labeling Kit (TaKaRa) with [α‐32P]dCTP. PCR primers are listed in Table S3 . Hybridization was carried out in Perfect Hyb Plus hybridization buffer (Sigma‐Aldrich, St. Louis, MO, USA) containing 32P‐labelled DNA probe at 50°C for 6 h. Membranes were washed four times in 2× SSC (1× SSC, 0.15 m NaCl, 15 mm sodium citrate) with 0.5% SDS at 50°C for 1 h, and then analyzed by a Typhoon FLA 7000 image analyzer (GE Healthcare, Chicago, IL, USA) (Fukuhara et al, 2011 ).
6
Dual-Incision Assay for (6-4) Photoproduct
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Dual-incision assays (Supplementary Fig. S1d ) were performed according to the described method35 (link), using the 32P-labeled 140 bp duplexes with and without the (6–4) photoproduct (35 fmol) and the whole cell extracts36 (link) in 50 μl solutions for 2 h, followed by 14% denaturing PAGE and band detection with a Typhoon FLA 7000 image analyzer (GE Healthcare). Fluorescence intensities (Supplementary Fig. S1e ) were measured at 37°C, using the same assay solutions after dilution to 200 μl with 1 M Tris-HCl (pH 8.0), on a JASCO FP-6500 spectrofluorometer equipped with an EHC 573 temperature controller. The excitation and emission wavelengths were 494 and 520 nm, respectively, with bandwidths of 3 nm. For the transfection of the XP12ROSV cells25 (link) (Supplementary Fig. S1f ), the cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2 incubator. The cells were seeded into a 4-well slide lumox (Greiner Bio-One), grown to about 90% confluence, and transfected with the 140 bp duplexes using Lipofectamine 2000 (Life Technologies), according to the manufacturer's instructions. At 6 h after transfection, the cells were analyzed with an Olympus IX71 fluorescence microscopy system.
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