Total RNAs were isolated using TRIzol and then subjected to a two-step purification to obtain tRNA. First, a PureLink RNA Mini extraction kit (Life Technologies) was used to precipitate the ribosomal RNA in the sample. In the second step, size-exclusion chromatography (SEC-HPLC) was performed using an Agilent Bio SEC-3 column with 10 mM ammonium acetate at pH 7 as the mobile phase. Neutral pH was selected to minimize hydrolysis of ct6A. tRNA and rRNA were collected as separate fractions, vacuum concentrated and reconstituted in water. tRNA concentrations were determined by UV spectroscopy at 260 nm. In the yeast samples, the proportion of tRNA to ribosomal RNA was much higher so only SEC-HPLC fractionation was required to obtain pure tRNA for analysis.
Sec 3 column
Manufactured by Agilent Technologies
The SEC-3 column is a size exclusion chromatography (SEC) column designed for the separation and analysis of macromolecules, such as proteins and polymers. It facilitates the separation of analytes based on their size and molecular weight. The column's core function is to enable efficient and reliable size-based separation and characterization of various samples.
Automatically generated - may contain errors
6 protocols using sec 3 column
1
Purification of tRNA from Total RNA
2
Small-Angle X-Ray Scattering Analysis of osTSN
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Small-angle X-ray scattering data were recorded at 15°C at the SAXS beamline 23A NSRRC in Hsinchu, Taiwan. The osTSN protein sample (1 mg/mL in PBS buffer containing an additional 150 mM NaCl and 5 mM β-mercaptoethanol at pH 7.4) was injected into an Agilent-Bio SEC-3 column at a flow rate of 0.02 mL/min. An X-ray wavelength of 0.8266 Å was used for data collection. Selected frames were merged and analyzed for initial Rg estimation by the PRIMUS program (Konarev et al. 2003 ), and then by the GNOM program (Svergun 1992 ) for Dmax and P(r) distance distribution (ATSAS program suite, version 2.7) (Petoukhov et al. 2012 (link)). Computation of the ensemble of osTSN was conducted using the Ensemble Optimization Method using ATSAS online (Tria et al. 2015 ).
3
Conjugation of 3E8.scFv.Cys with IRDye800-maleimide
4
Characterization of n501-αHSA Antibody
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SDS‐PAGE was used to detected the size of n501–αHSA and high‐performance liquid chromatography (SEC‐HPLC) was used to determine the uniformity of n501–αHSA. For SDS‐PAGE, SmartPAGE™ 4−12% Bis‐Tris Protein precast gels (Smart Life‐Science, China) were used for SDS‐PAGE analysis, and approximately 5 µg of antibodies were loaded per well. After electrophoresis at 160 V for 40 min, the gel was stained with Coomassie Blue Fast Staining buffer (Beyotime Biotechnology, China) and destained with running water.
For SEC‐HPLC analysis, n501–αHSA was diluted to 1 mg/mL and 10 µL was added onto an Agilent Bio SEC‐3 column (150 Å, 3 µm, 4.6 × 150 mm) at 0.5 mL/min. PBS buffer was used as the mobile phase. The absorption peak at 280 nm was obtained to detect the uniformity of the antibody.
For SEC‐HPLC analysis, n501–αHSA was diluted to 1 mg/mL and 10 µL was added onto an Agilent Bio SEC‐3 column (150 Å, 3 µm, 4.6 × 150 mm) at 0.5 mL/min. PBS buffer was used as the mobile phase. The absorption peak at 280 nm was obtained to detect the uniformity of the antibody.
5
SAXS Analysis of TrmJ C-Termini
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All SAXS measurements were performed at 15°C on the SWING beamline (SOLEIL, France). An inline size-exclusion chromatography set-up was used prior to data collection. Here, 50 µL of SaTrmJ163-235 or EcTrmJ167-246 at 8 mg/mL was injected on an Agilent Bio SEC-3 column (300 Å pore size), which ran at 0.2 mL/min in 20 mM Tris (pH 8), 150 mM NaCl buffer. Data processing was done using the ATSAS package (Petoukhov and Svergun 2013 (link)). The multimerization state of these C-terminal constructs was determined through the SAXSMoW application (Fischer et al. 2010 ).
6
Protein Purification and Analysis
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Affinity purified proteins or indicated in vitro reactions were resolved on an Agilent 1100 HPLC system equipped with an Agilent SEC-3 column in the purification or in vitro reaction buffer.
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