The DNA template of each tRNA was generated using the Primerize method (Tian et al., 2015 (link)) and the T7 promoter sequence was included for T7 RNA polymerase driven in vitro transcription (T.‐Y. Lin et al., 2019 (link)). An overnight T7‐driven transcription reaction was performed at 37°C with the following: 20 mM Tris, pH 8.0, 5 mM DTT, 150 mM NaCl, 8 mM MgCl2, 2 mM spermidine, 20 mM NTPs, linear DNA, RNasin, T7 RNA polymerase, and pyrophosphatase. RNase‐free DNase I (Thermo Fisher Scientific) was added to digest the DNA template and the reaction was stopped by the addition of EDTA. The reaction was subjected to a FPLC system using a DEAE weak anion exchange column (GE) and followed by temperature‐gradient‐based annealing. Refolding of RNA was carried out by heating the RNA solution to 80°C for 2 min and slowly cooling to room temperature. The annealed RNAs were further purified using a Superdex 75 Increase gel filtration column (GE) in the buffer (20 mM HEPES, pH 7.5, 5 mM DTT, 150 mM NaCl) and the fractions of interest were pooled, concentrated, and stored at −20°C.
Superdex 75 increase gel filtration column
Manufactured by GE Healthcare
Sourced in United States
The Superdex 75 Increase gel filtration column is a laboratory equipment used for size-exclusion chromatography. It is designed to separate molecules based on their size and molecular weight. The core function of this column is to facilitate the purification and analysis of proteins, peptides, and other biomolecules.
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2 protocols using superdex 75 increase gel filtration column
1
Purifying and Refolding RNA via FPLC and Annealing
2
Purification and Characterization of PvQ Proteases
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PvQ is a significant fibrinolytic enrichment, containing several active protein monomers. In order to clarify the effective substances of PvQ more thoroughly, a systematic study of its proteases in the fraction was performed in this project and a series of chromatographic techniques were used for purification [12 (link)].
Firstly, the lyophilized powders of PvQ were redissolved in 20 mM Tris-HCl buffer (pH 7.8) and purified on a HiTrap Q HP column (GE Healthcare, Chicago, IL, USA) eluted with a NaCl gradient from 0 to 20 mM at a flow rate of 5.0 mL/min. The fractions were pooled and assayed by the fibrin plate to track the active protein peak. The eluted active fractions were successively subjected to a HiPrep Phenyl FF column (GE Healthcare, Chicago, IL, USA), eluting with a stepwise gradient of 20 mM Tris-HCl buffer with 1 M ammonium sulfate (from 1 to 0.5 M, pH 7.8). Subsequently, size exclusion chromatography using a Superdex 75 Increase gel filtration column (GE Healthcare, Chicago, IL, USA) was carried out for each active HIC fraction through an AKTA pure protein purification system with 20 mM Tris-HCl buffer containing 20 mM NaCl at a flow rate of 0.5 mL/min. All columns were used repeatedly until SDS-PAGE showed single bands. Active fractions were pooled, desalted, and lyophilized.
Firstly, the lyophilized powders of PvQ were redissolved in 20 mM Tris-HCl buffer (pH 7.8) and purified on a HiTrap Q HP column (GE Healthcare, Chicago, IL, USA) eluted with a NaCl gradient from 0 to 20 mM at a flow rate of 5.0 mL/min. The fractions were pooled and assayed by the fibrin plate to track the active protein peak. The eluted active fractions were successively subjected to a HiPrep Phenyl FF column (GE Healthcare, Chicago, IL, USA), eluting with a stepwise gradient of 20 mM Tris-HCl buffer with 1 M ammonium sulfate (from 1 to 0.5 M, pH 7.8). Subsequently, size exclusion chromatography using a Superdex 75 Increase gel filtration column (GE Healthcare, Chicago, IL, USA) was carried out for each active HIC fraction through an AKTA pure protein purification system with 20 mM Tris-HCl buffer containing 20 mM NaCl at a flow rate of 0.5 mL/min. All columns were used repeatedly until SDS-PAGE showed single bands. Active fractions were pooled, desalted, and lyophilized.
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