Q sepharose ff

The plasmid pPIC9K and the yeast cell strain GS115 were obtained from the Key Laboratory of Molecular Medicine of Fudan University [15 (link)]. The E. coli strain DH5α was obtained from TIANGEN Biotech Co., Ltd. (Beijing).
Yeast nitrogen base (with or without amino acids) was purchased from Sigma. Q-Sepharose-FF and Sephadex G-50 were purchased from GE Healthcare.

The sources of the chemicals were described previously [16 (link)]. Leaves from elder were harvested at Cobos de Cerrato (Palencia, Spain) in early summer. CM-Sepharose FF, Q-Sepharose FF, CM-Sepharose FF, Sepharose 6 B, and Superdex−75 HiLoad 26/60 columns were purchased from GE Healthcare (Barcelona, Spain). The acid-treated Sepharose (AT-Sepharose) was prepared as described in [66 (link)], treating the Sepharose 6 B with 0.1 N HCl at 50 °C for 3 h. The gel was then washed with water (Elix 5, Millipore) until a neutral pH was obtained, and stored in water at 4 °C until it was used. Tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin was purchased from Merk Life Science S.r.l. (Milan, Italy). Acetonitrile (CH3CN), formic acid (FA), and water (LC–MS grade) were from Fisher Scientific Italia (Milan, Italy). Century™-Plus RNA Markers were purchased from Fisher Scientific (Madrid, Spain). Rabbit reticulocyte lysate system (nuclease-treated) was purchased from Promega Biotech Iberica S.L. (Alcobendas, Madrid, Spain).

E. coli strain MG1655 was transformed with pBAD-AtaT or empty pBAD/Myc-His A (Invitrogen) and cultured overnight. The overnight cultures were diluted to an OD₆₆₀ of 0.02 in fresh liquid LB (4 mL) containing 50 μg/ml ampicillin, and cultured at 37 °C until the A₆₆₀ reached 0.2. Then, 0.02% (w/v) arabinose was added. After a 15, 30, or 60 min incubation, the cells were harvested and suspended in buffer containing 50 mM NaOAc, pH 5.0, 0.5 mM EDTA, and 0.2 M NaCl. RNA was extracted by phenol saturated with 300 mM NaOAc, pH 5.2, followed by isopropyl alcohol precipitation. The RNA was dissolved in 250 μL of 200 mM NaOAc, pH 5.0, acetylated by adding acetic anhydride-D₆ (Sigma Aldrich, Japan)^{38 (link)}, and then ethanol precipitated and rinsed with 70% cold ethanol. The RNA was dissolved in cold buffer containing 50 mM NaOAc, pH 5.0, 0.5 mM EDTA, and 0.2 M NaCl, and loaded onto a 100 μL Q-Sepharose F.F. (GE Healthcare, Japan) column. The resin was washed with buffer containing 50 mM NaOAc, pH 5.0, 0.5 mM EDTA, and 0.2 M NaCl. tRNA was eluted with buffer containing 50 mM NaOAc, pH 5.0, 0.5 mM EDTA, and 0.6 M NaCl, ethanol precipitated, and rinsed with 70% (v/v) ethanol.