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Fla 3000

Manufactured by Fujifilm
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The FLA-3000 is a fluorescence imaging analyzer designed for life science applications. It is capable of detecting and analyzing fluorescent signals from various samples, including gels, membranes, and microplates.

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Lab products found in correlation

γ 32p atp, by PerkinElmer (8 mentions) T4 polynucleotide kinase, by New England Biolabs (6 mentions) Amersham hybond n, by GE Healthcare (2 mentions) Bas 5000, by Fujifilm (2 mentions) Whole genome amplification, by Merck Group (2 mentions) Quantity one software, by Bio-Rad (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Thermopol buffer, by New England Biolabs (1 mentions) Rnase hii, by New England Biolabs (1 mentions) Tbe urea gel, by Thermo Fisher Scientific (1 mentions) Perfecthyb plus hybridization buffer, by Merck Group (1 mentions) Herring sperm dna, by Thermo Fisher Scientific (1 mentions) Hybond n membrane, by GE Healthcare (1 mentions) Nylon membrane, by Cytiva (1 mentions) Pei cellulose f plate, by Merck Group (1 mentions) Decalabel dna labeling kit, by Thermo Fisher Scientific (1 mentions) Vacuum gel drier, by Bio-Rad (1 mentions) Fusion sl imaging system, by Vilber (1 mentions) Microtubule binding protein spin down assay kit, by Cytoskeleton (1 mentions) Qiaquick nucleotide removal kit, by Qiagen (1 mentions)

Close spelling variants of this product

Fuji FLA-3000 phosphoimager Image Analyzer FLA-3000 FLA-3000 phosphoimager FLA 3000 plus DAGE system FLA-3000 laser fluorescence scanner FLA-3000 Series FLA-3000 Phosphor Screen FLA-3000 system FLA-3000 scanner Fuji FLA-3000 Phosphor-Imager

66 protocols using fla 3000

1

Isolation and Quantification of Radiolabeled rRNA

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We followed the RNAsnap procedure (46 (link)) to isolate rRNA from 32P labelled exponential cultures (OD600 approximately 0.3) grown at 37°C. The rRNA samples were precipitated out via ethanol reprecipitation (0.1 × 5 M NaCl and 2× ethanol were added to 1× volume of rRNA sample and mixed vigorously by vortexing. The samples were then spun down at 16 000g for 5 min, and the supernatant was completely removed via aspiration and the pellet was resuspended in TE) prior to running them on a 1.1% agarose gel at 60 V for 2 h. After the run, the gel was dried and exposed to a PhosphorImager screen. The resulting signals were measured with a PhosphorImager (Fuji Film FLA-3000).
Mahaseth T, & Kuzminov A. (2022). Catastrophic chromosome fragmentation probes the nucleoid structure and dynamics in Escherichia coli. Nucleic Acids Research, 50(19), 11013-11027.
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2

In Vitro Phosphorylation Kinetics of Cyanobacterial KaiC Proteins

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To investigate KaiA dependent phosphate uptake 12 μg of KaiC-7942, KaiC1-Sy6714, KaiC1-N29133, KaiC1-Cy7424, KaiC3-Cy7424, KaiC3-Mic7806, KaiC3-T.lit or KaiC3-P.hor were mixed with 10 μCi γ-P32-ATP in 60 μl Tris reaction buffer (20 mM Tris/HCl (pH 8), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 1 mM ATP) in the presence or absence of 6 μg KaiA-7942. 10 μl aliquots were taken after 0, 0.75, 1.5, 3 and 22 hours of incubation at 30 °C and reaction was stopped by adding SDS-sample buffer. Proteins were separated in high-resolution polyacrylamide gels (10% T, 0.67% C) by SDS-PAGE (modified from [87 (link)]), stained with Coomassie brilliant blue and subjected to autoradiography. Signals were analyzed using a Fujifilm FLA-3000 (FUJIFILM). To analyze in vitro phosphorylation of KaiC3-T.lit and KaiC3-P.hor at higher temperatures, 10 μg of the recombinant proteins were incubated with 10 μCi γ-P32-ATP in 50 μl HEPES reaction buffer (50 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM MgCl2, 1 mM ATP) or 50 μl MES reaction buffer (50 mM MES (pH 6), 150 mM NaCl, 5 mM MgCl2, 1 mM ATP), respectively, at 75 °C. After 0, 5, 10 and 15 minutes 10 μl aliquots were taken and analyzed by SDS-PAGE and autoradiograpy as described above. Comprehensive protocols are available on protocols.io (dx.doi.org/10.17504/protocols.io.g3gbyjw, dx.doi.org/10.17504/protocols.io.gysbxwe).
Schmelling N.M., Lehmann R., Chaudhury P., Beck C., Albers S.V., Axmann I.M, & Wiegard A. (2017). Minimal tool set for a prokaryotic circadian clock. BMC Evolutionary Biology, 17, 169.
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3

Detecting RNA-DNA Intermediates in Plasmids

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50 or 500 ng of purified DNA (total plasmid, or alkaline lysis, or total genomic preparation) were incubated in 20 μl of 1× Thermopol buffer (NEB) containing either no enzyme or 1.5-2.5 units of RNase HII (NEB). DNA was incubated with 2.5 units of RNase HI (Ambion or ThermoScientific) in 20 μl of 1× RNase HI buffer (ThermoScientific): 20 mM Tris HCl (pH 7.8), 40 mM KCl, 8 mM MgCl2, 1 mM DTT. Incubation was at 37°C for 30 minutes. After incubation the reaction mixtures were chilled on ice and directly loaded on 1.1 % agarose gels and run in 1×TAE buffer.
To remove potential (RNA-DNA) intermediates formed in plasmids, the DNA samples were treated in 30 μl of 70% formamide at 65°C for 5 minutes, chilled on ice, precipitated with 500 mM NaCl and ethanol, dried on air and dissolved in water and aliquotted into three 20 μl reactions for treatment with RNase HI or RNase HII.
DNA species were analyzed by Southern hybridization, the radioactive membranes were scanned by PhosphorImager (FujiFilm FLA-3000, Fuji). For calculation of DNA representing zero class of the Poisson distribution by the enzymatic method, radioactivity in supercoiled monomer band was divided by the total radioactivity in the lane area between the relaxed monomer band and the supercoiled monomer band.
Kouzminova E.A., Kadyrov F.F, & Kuzminov A. (2017). RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation. Journal of molecular biology, 429(19), 2873-2894.
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4

In Vitro Organelle Protein Import Assay

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Radiolabeled precursor proteins of AtWHIRLY3 and the organelle‐specific control proteins FNR (chloroplast Ferredoxin‐NADP‐Oxidoreductase) and mitochondrial Rieske Fe/S protein were obtained by in vitro translation in rabbit reticulocyte lysates in the presence of [35S]‐methionine. Incubation with intact mitochondria or chloroplasts isolated from pea leaves followed the protocol of Rödiger, Baudisch, and Bernd Klösgen (2010). Competition experiments were performed as described (Bennewitz, Sharma, Tannert, & Klösgen, 2020).
Gel electrophoresis of proteins under denaturing conditions was carried out according to Laemmli (1970). The gels were exposed to phosphorimaging screens and analyzed with a Fujifilm FLA‐3000 (Fujifilm) using the software packages BAS Reader (version 3.14) and AIDA (version 3.25; Raytest). Protein concentration was determined according to Bradford (1976).
Golin S., Negroni Y.L., Bennewitz B., Klösgen R.B., Mulisch M., La Rocca N., Cantele F., Vigani G., Lo Schiavo F., Krupinska K, & Zottini M. (2020). WHIRLY2 plays a key role in mitochondria morphology, dynamics, and functionality in Arabidopsis thaliana. Plant Direct, 4(5), e00229.
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5

Optimization of DNA Ribonucleotide Cleavage

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To find optimal temperature, molarity of NaOH and time of treatment to cleave a single ribonucleotide embedded in DNA, we treated 0.5 pmole of 5′ end-radiolabeled 38-R1 oligonucleotide annealed to the complementary DNA template substrate mixed with 6 ng of cold 2 kbp DNA oriC-fragment in either 10 or 20 μl reactions containing NaOH and 5 mM EDTA. The concentrations of NaOH varied from 0.1 to 0.5 M, the incubation time varied from 30 minutes to overnight, the tested temperatures were: 0, 4, 16, 25, 37, 45 and 55°C. After the reaction, half of the sample was analyzed on 12% native polyacrylamide gel (to test rNMP hydrolysis), while the other half was run on 0.7% agarose in 1×TAE gel followed by Southern analysis with the oriC-probe to test the stability of DNA. The polyacrylamide gel was directly scanned by PhosphorImager (FujiFilm FLA-3000, Fuji). The extent of hydrolysis was estimated by taking the ratio of radioactivity present in the cleaved product relative to the total signal (both the uncleaved and the cleaved products together).
Kouzminova E.A., Kadyrov F.F, & Kuzminov A. (2017). RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation. Journal of molecular biology, 429(19), 2873-2894.
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6

Northern Blot Analysis of RNA

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Northern blot analysis was performed using 1 μg of total RNA on a 10% TBE–urea gel (Thermo Fisher). The RNA was transferred to a Hybond-N+ membrane (GE Healthcare) post UV crosslinking. The membrane was dried and pre-hybridized at 68 °C for 30 min in PerfectHyb plus hybridization buffer (Sigma) and 0.1 mg ml−1 herring sperm DNA (Thermo Fisher). During hybridization, 1 × 106 c.p.m. ml−1 of 32P-labelled LNA/DNA probe was added2 (link). The membrane was hybridized at 68 °C overnight. Subsequently, the membrane was washed once in low stringency buffer (2×SSC and 0.1% SDS) at room temperature for 5 min and twice in high stringency buffer (0.5×SSC and 0.1% SDS) at 68 °C for 20 min. Quantification was performed overnight using a phosphorimager (Fuji film FLA3000). After exposure, the membranes were incubated in boiling stripping buffer (0.1% SDS and 5 mM EDTA) and blotted with U6 probe for the loading control.
Guzzi N., Muthukumar S., Cieśla M., Todisco G., Ngoc P.C., Madej M., Munita R., Fazio S., Ekström S., Mortera-Blanco T., Jansson M., Nannya Y., Cazzola M., Ogawa S., Malcovati L., Hellström-Lindberg E., Dimitriou M, & Bellodi C. (2022). Pseudouridine-modified tRNA fragments repress aberrant protein synthesis and predict leukaemic progression in myelodysplastic syndrome. Nature Cell Biology, 24(3), 299-306.
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7

Determination of SOMAmer Binding Affinity

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Example 8

For determination of target binding affinity, SOMAmers were 5′ end-labeled using T4 polynucleotide kinase (New England Biolabs) and γ-32P-ATP (Perkin Elmer). Binding assays were performed by incubating radiolabeled SOMAmer (˜20,000 c.p.m) at a concentration of ˜0.03-0.05 nM and target protein at concentrations ranging from 10−7 to 10−12 M in 1×SB18T buffer (40 mM HEPES, pH 7.5; 120 mM NaCl; 5 mM KCl; 5 mM MgCl2 and 0.01% TWEEN-20) at 37° C. for 30 minutes. Bound complexes were mixed with Zorbax resin and captured on Durapore filter plates. The fraction of SOMAmer bound was quantified with a PhosphorImager (FUJI FLA-3000). Raw binding data were corrected for nonspecific background binding of radiolabeled SOMAmer to Zorbax resin. Equilibrium dissociation constants (Kd) was determined as previously described (Jellinek et al. (1993) Proc. Natl. Acad. Sci. 90:11227).

US10544419B2. PDGF and VEGF aptamers having improved stability and their use in treating PDGF and VEGF mediated diseases and disorders (2020-01-28). SomaLogic, Inc. [US]. Inventors: Nebojsa Janjic [US], Daniel W. Drolet [US], Amy D. Gelinas [US], Chi Zhang [US], Michael Vrkljan [US].
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8

DNA Extraction and Origin-Terminus Ratio

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Total DNA was extracted from saturated and exponentially growing cultures by the phenol::chloroform method (Kouzminova & Kuzminov, 2006 (link)). 1 μg was denatured in 400 μl 0.1 M NaOH for 15 minutes at 37°C and spotted in duplicate on a positively charged Nylon membrane (Amersham) using a vacuum manifold. After cross-linking, the membrane was divided in two with one half hybridizing to the origin-proximal probe and the other half to the terminus-proximal probe (Kouzminova & Kuzminov, 2006 (link)). The spot intensity was measured using the PhosphorImager (FujiFilm FLA-3000), and the ori/ter value was calculated by normalizing to the ori/ter value of a saturated overnight AB1157 culture, which was set to 1.0.
Rotman E., Khan S., Kouzminova E, & Kuzminov A. (2014). Replication fork inhibition in seqA mutants of E. coli triggers replication fork breakage. Molecular microbiology, 93(1), 50-64.
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9

Agarose Plug Hybridization for Ori/Ter Ratio

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L-110 and AB1157 strains were grown overnight in LB at 43°C. Next morning, the cultures were diluted to OD 0.1 and grown at 28°C with shaking. 1 ml aliquots of the growing cultures were taken out at specified ODs to make two agarose plugs. The procedure for making, treating agarose plugs and for agarose plug hybridization was as described (Kouzminova & Kuzminov, 2012 ). 32P-labelled PCR amplified fragments containing oriC and dif chromosomal regions were used for hybridization and described (Kouzminova & Kuzminov, 2008 (link)). The signals from the plugs at various ODs were measured with the Phosphorimager (FujiFilm FLA-3000) and normalized to the signal of the plug with OD 0.1. The ori/ter ratios in Fig. 5I were derived from the signals used to calculate ori and dif copy number increase in panels G and H.
Rotman E., Khan S., Kouzminova E, & Kuzminov A. (2014). Replication fork inhibition in seqA mutants of E. coli triggers replication fork breakage. Molecular microbiology, 93(1), 50-64.
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10

Pulsed-field gel analysis of DNA

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Non-radioactive plugs were made and run in duplicate on pulsed field gels as above. After the gel was finished, it was treated for capillary transfer to nylon membrane (Hybond H+). Due to the thickness of the gel, before transfer the plugs were removed and laid on their side. The membrane was divided in two and prehybridized in 5% SDS, 0.5M sodium phosphate pH 7.4 at 65°C for 1 hour. One half of the membrane was probed with 32P-labeled origin-specific probe, while the other half was probed with the terminus-specific probe (Kouzminova & Kuzminov, 2006 (link)). Hybridization in the same conditions was overnight; after its completion, the membranes were washed three times in 1/10 strength prehybridization buffer before exposure and quantification by PhosphorImager (FujiFilm FLA-3000).
Rotman E., Khan S., Kouzminova E, & Kuzminov A. (2014). Replication fork inhibition in seqA mutants of E. coli triggers replication fork breakage. Molecular microbiology, 93(1), 50-64.
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