Transfections of HEK293T cells were performed as described above. Briefly, 3 µg plasmid DNA (EGFP-MmDHX37wt and EGFP-MmDHX37ΔCTD) were transfected in HEK293T cells at a density of 2 × 106 cells using 9 µL of the X-tremeGENE HP DNA Transfection Reagent (Roche). Forty-eight hours posttransfections, cells were harvested and lysed in 1.5 mL assay buffer (20 mM Tris pH 7.5, 150 mM KCl, 1 mM EDTA, 0.1% [v/v] Tween20, 1 mM DTT) supplemented with Roche Protease Inhibitor Cocktail, in the presence of RNAse A (Thermo Fisher). Per reaction, 50 µL (packed volume) Amylose beads (NEB) were incubated with 2 mg of recombinant His6-MBP-HsUTP14Amin for 30 min at 4°C and washed three times with HEK assay buffer. For the pull-downs, 500 µL of cleared HEK293T cell lysates were incubated with 50 µL His6-MBP-HsUTP14Amin-Amylose-beads for 1 h at 4°C. For the no bait control, 50 µL Amylose-beads without His6-MBP-HsUTP14A555–619 were incubated with 500 µL HEK293T cell lysate. The beads were washed four times with HEK293T assay buffer and bound proteins were eluted in 1× SDS-PAGE sample loading dye supplemented with 10 mM maltose. Proteins were separated by SDS-PAGE with no prior boiling to avoid EGFP denaturation, visualized on a Typhoon FLA9500 fluorescence scanner (GE Healthcare) and subsequently stained with Coomassie brilliant blue R250.
Typhoon fla 9500 fluorescence scanner
Manufactured by GE Healthcare
Sourced in United States
The Typhoon FLA 9500 is a fluorescence scanner designed for high-performance imaging of a wide range of sample types, including gels, microarrays, and blots. It utilizes laser-based excitation and sensitive photomultiplier tube (PMT) detection to capture detailed images of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Amylose beads, by New England Biolabs (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Strep tactin sepharose beads, by GE Healthcare (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
5 protocols using typhoon fla 9500 fluorescence scanner
1
EGFP-tagged MmDHX37 Pulldown with HsUTP14A
2
Visualization of OA-DNA Complexes
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OA–DNA complexes were formed in the binding buffer (20 mM Tris-HCl pH 8.0, 50 mM KCl) with 500 nM of DNA fragments and indicated OA concentrations for 20 min at 25 °C. Samples were resolved in a 8% non-denaturing polyacrylamide gel electrophoresis and visualized on a Typhoon FLA 9500 fluorescence scanner (GE Healthcare, Chicago, IL, USA) equipped with a 473 nm blue LD laser and LBP filter.
3
Affinity Purification of CPSF Complexes
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For pull-down assays from Sf9 cells expressing CPSF subunits, the cells were resuspended in 20 mM Tris-Cl pH 7.5, 200 mM NaCl, 10% glycerol and 0.05% Tween20, and lysed by sonication. The clarified lysate was incubated with 600 μl of NiNTA resin (Qiagen). After washing, the protein was eluted and incubated directly with 30 μl Streptactin sepharose beads (GE Healthcare). The beads washed three times with 1 ml of resuspension buffer and the protein eluted with SDS-PAGE loading buffer at room temperature. The eluted samples and the input lysate were loaded on SDS-PAGE with no prior boiling to avoid GFP denaturation, visualized on a Typhoon FLA9500 fluorescence scanner (GE Healthcare) and subsequently stained with Coomassie brilliant blue R250.
For pull-down assays with purified proteins, 3 μg of GST-Fip1CD was immobilized on glutathione sepharose four fast flow beads (GE Healthcare) and incubated with 40 μg of MBP-CPSF30. The beads were washed three times with 500 μl of buffer containing 20 mM Tris-Cl pH 7.5, 150 mM NaCl and 0.05% Tween20, the sample eluted with SDS-PAGE loading buffer and analyzed by SDS-PAGE.
For pull-down assays with purified proteins, 3 μg of GST-Fip1CD was immobilized on glutathione sepharose four fast flow beads (GE Healthcare) and incubated with 40 μg of MBP-CPSF30. The beads were washed three times with 500 μl of buffer containing 20 mM Tris-Cl pH 7.5, 150 mM NaCl and 0.05% Tween20, the sample eluted with SDS-PAGE loading buffer and analyzed by SDS-PAGE.
4
Plasmid Construction for Split-GFP Assay
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Construction of plasmids and expression for split-GFP reassembly were performed according to published protocols (29 (link)). pET11a-NGFP constructs encoding E. coli ProRS and E. coli YbaK sequences (NGFP-ProRS and NGFP-YbaK) and a pMRBAD-CGFP construct encoding E. coli ProRS (CGFP-ProRS) were prepared using standard PCR cloning strategies. Plasmid sequences were confirmed by DNA sequencing. The plasmids encoding pET11a-Z-NGFP (NGFP-zipper), pMRBAD-Z-CGFP (CGFP-zipper), and pMRBAD-link-CGFP (CGFP-linker) were constructed as described (30 (link)). E. coli BL21 (DE3) cells were cotransformed with the following pairs of plasmids: (I) NGFP-zipper and CGFP-zipper; (II) NGFP-ProRS and CGFP-ProRS; (III) NGFP-YbaK and CGFP-YbaK; (IV) NGFP-YbaK and CGFP-ProRS; (V) NGFP-YbaK and CGFP-linker; (VI) NGFP-ProRS and CGFP-linker. Single colonies were selected and grown in LB media containing 100 μg/ml ampicillin and 35 μg/ml kanamycin at 37°C overnight. Overnight cultures were diluted 1000-fold and 10-μl aliquots were spread on 2× YT agar plates containing 100 μg/ml ampicillin, 35 μg/ml kanamycin, 10 μM IPTG and 0.2% arabinose. The plates were incubated at room temperature for 36 h. Plate images were obtained with a Typhoon FLA 9500 fluorescence scanner (GE Healthcare) equipped with 473 nm blue LD laser and 575 nm LPG filter.
5
Visualizing OA-DNA Complexes by Fluorescence
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Non-labeled DNA hairpins at low (50 nM) or high (2.5 µM) concentrations (indicated in figure legends) were pre-incubated for 30 min with OA in buffer A containing 100 mM NaCl, 5 mM MgCl2 and 20 mM Tris pH 8.0 before loading on a 10% PAAG. Electrophoresis was run for 1–2 h at 15 V/cm, either in a 45 mM Tris base/45 mM boric acid/1 mM EDTA (0.5× TBE buffer) pH 8.3 or in 0.5× TBM buffer pH 8.3 which contained 1 mM magnesium acetate instead of EDTA. The OA–DNA complexes at the high concentration were directly visualized by intrinsic fluorescence on a ChemiDoc™ XRS+ System (BioRad, Hercules, CA, USA) after UV excitation of the bound drug. Then, the same gels were stained with ethidium bromide (EtBr) for 15 min to detect DNA. The OA–DNA complexes formed at the low DNA concentration were visualized by staining the gel with SybrGold and scanning in the Typhoon FLA 9500 fluorescence scanner (GE Healthcare, Chicago, IL, USA) equipped with a 473-nm blue LD laser and LBP filter.
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