To examine the interactions of MRS221–900 and the MRS–AIMP3 complex with tRNA, pull-down assays were performed using Ni-NTA resin. Yeast total tRNA (Sigma-Aldrich) was subjected to a pull-down assay with hexa-His-tagged proteins immobilized on Ni-NTA resin. A 2.5 nmol aliquot of each protein was bound to Ni-NTA resin and washed several times with buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl). The pull-down assay was performed by adding 1.25 mg yeast tRNA, and resin was washed three times with buffer. The protein and bound tRNA were eluted with buffer containing 200 mM imidazole. UV absorption spectra of the eluted samples and buffers were measured by wavelength scanning from 320 to 230 nm using a UV-1800 UV spectrophotometer (Shimadzu). The amount of tRNA and protein was analyzed by measuring the absorbance at 260 and 280 nm. Synthesized RNA with an elongator tRNAMet sequence labeled with 6-carboxyfluorescein (6-FAM) was purchased from Integrated DNA Technologies and used for the pull-down assay. The amount of RNA eluted was estimated by fluorescence at wavelengths of 495 nm and 517 nm for excitation and emission, respectively.
6 carboxyfluorescein 6 fam
Manufactured by Integrated DNA Technologies
6-carboxyfluorescein (6-FAM) is a fluorescent dye used as a reporter molecule in various molecular biology applications. It emits a green fluorescent signal upon excitation, making it useful for labeling and detection of biomolecules such as nucleic acids and proteins.
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Lab products found in correlation
Uv spectrophotometer uv 1800, by Shimadzu (1 mentions)
Yeast total trna, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Guanosine 5 triphosphate gtp, by Thermo Fisher Scientific (1 mentions)
Tris hcl, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
2 protocols using 6 carboxyfluorescein 6 fam
1
Probing tRNA Interactions with MRS Complexes
2
ATP-Binding Aptamer Characterization Protocol
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Jurkat, Clone E6.1 (T lymphocyte), cells were purchased from the American Type Culture Collection (ATCC). The cell line was cultured in HyClone RPMI-1640 (+25mM HEPES, +L-Glutamine) medium supplemented with 100 units/mL penicillinstreptomycin 1% (Corning), 1% MEM Non-Essential Amino Acids (Gibco) and 10% fetal bovine serum (Heat Inactivated, Gibco). All cell lines were routinely evaluated on a Flow Cytometer (FACScan, Becton Dickinson) for the expression of CD marker using anti-hCD3ε (PE-conjugated Mouse IgG1, R&D Systems) antibody to authenticate the cell line. All conformational assays were tested using Adenosine-5'triphosphate, ATP, from a stock solution of 100 mM (ThermoFisher). The specificity assay was performed using 2 mM of Adenosine-5'-triphosphate, ATP, Uridine-5'triphosphate, UTP, Cytidine-5'-triphosphate, CTP, and Guanosine-5'-triphosphate, GTP, from a stock solution of 100mM (ThermoFisher). All aptamer solutions were prepared in 10 mM Tris-HCl (Thermo Scientific) adjusted to pH=8.4, with 6 mM MgCl2 (Sigma Aldrich) from a stock solution of 1M Tris-HCl (ThermoFisher). All DNA sequences were ordered HPLC-purified from Integrated DNA Technologies (IDT) and dual-modified with 6-Carboxyfluorescein (6-FAM) and Iowa Black Fluorescence Quencher (IBFQ) at the 5' and 3', respectively.
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