The expression plasmids for ttCK mutants were transformed into Escherichia coli Rosetta(DE3). The transformants were cultured at 37 °C in 1.5 L of LB medium supplemented with 50 μg mL−1 of ampicillin for 12 h. Cells were harvested by centrifugation. ttCK was purified by heat treatment at 70 °C for 20 min and column chromatography with Toyopearl Phenyl 650 M (Tosoh), Toyopearl SP-650 column (Tosoh), and Superdex 200 10/300 GL column (GE Healthcare Biosciences). The expression plasmid for GST-fusion hsUCK2 (wild type (WT)) and its mutant (H117Y) were transformed into E. coli BL21(DE3). The transformants were cultured at 37 °C in 1.5 L of LB medium supplemented with 50 μg mL−1 of ampicillin for 12 h. Cells were harvested by centrifugation. hsUCK2 was purified by the column chromatography with GSTrap FF (GE Healthcare Biosciences). The concentrations of the purified ttCK variants were determined by using molar absorption coefficients at 278 nm, calculated to be 11,200 for ttCK variants except ttCK (Y93W) and 16,800 M−1 cm−1 for ttCK (Y93W) [11] (link).
Toyopearl phenyl 650 m
Manufactured by Tosoh
Sourced in Japan
Toyopearl Phenyl 650 M is a chromatography resin designed for protein purification. It features a hydrophobic phenyl ligand covalently bonded to a porous agarose matrix. The resin is suitable for use in reversed-phase liquid chromatography applications.
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Lab products found in correlation
Gstrap ff, by GE Healthcare (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Hitrap q hp column, by GE Healthcare (1 mentions)
Kta prime chromatography system, by GE Healthcare (1 mentions)
Hiload superdex 200 pg, by GE Healthcare (1 mentions)
Glass econo column, by Bio-Rad (1 mentions)
Vivaspin 20, by Sartorius (1 mentions)
Toyopearl deae 650m, by Tosoh (1 mentions)
5 protocols using toyopearl phenyl 650 m
1
Purification of Recombinant Kinase Enzymes
2
Purification of Recombinant Protein by FPLC
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The first step of protein purification by anion-exchange chromatography was performed as described previously (Ishibashi et al., 2005 ▶ , 2011 ▶ ). The soluble fraction of disrupted cells was applied onto a HiTrap Q HP column (1.6 × 2.5 cm, GE Healthcare) using an ÄKTAprime chromatography system (GE Healthcare). The bound proteins were eluted with a 100 ml linear gradient of NaCl from 0.3 to 0.9 M in 50 mM Tris–HCl pH 8.0, 2 mM MgCl2 buffer. The fractions containing HaAP were pooled and dialyzed against 50 mM Tris–HCl pH 8.0, 2 mM MgCl2 containing 3 M NaCl buffer. After dialysis, 30% ammonium sulfate was added and the soluble fraction was collected by centrifugation at 12 000g for 15 min. Proteins in the soluble fraction were loaded onto a hydrophobic column (30 ml, Toyopearl Phenyl-650M, Tosoh Bioscience) equilibrated with 50 mM Tris–HCl pH 8.0 containing 2 mM MgCl2, 0.5 M NaCl, 30% ammonium sulfate. The flowthrough fraction was then applied onto a gel-filtration column (1.6 × 60 cm, HiLoad Superdex 200 pg, GE Healthcare) equilibrated with 50 mM Tris–HCl pH 8.0, 2 mM MgCl2 buffer containing 0.5 M NaCl. The elution was pumped at 0.5 ml min−1. The protein purity was checked by 10% SDS–PAGE.
3
Purification of GAA from Arabidopsis alg3
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The medium of GAA-producing Arabidopsis alg3 culture was filtered through a Glass Econo-Column (Bio-Rad). NaCl was subsequently added to the medium until reaching a final concentration of 4 M. Then, the NaCl-containing medium was loaded into a hydrophobic interaction chromatography column (Toyopearl Phenyl-650 M, Tosoh Corporation) pre-equilibrated in 20 mM Tris-HCl pH 7.5 with 4 M NaCl. After washing the column using 4 M NaCl in 20 mM Tris-HCl pH 7.5 buffer, GAA was eluted by decreasing NaCl concentration. GAA-containing fractions were dialyzed to exchange the buffer into 20 mM sodium acetate pH 4.3. The dialyzed sample was applied to a cation exchange chromatography column (Toyopearl SP 550C, Tosoh Corporation) pre-equilibrated in 20 mM sodium acetate pH 4.3 buffer. After washing the column, GAA was eluted with 20 mM sodium acetate pH 4.3 containing 0.1–0.5 M NaCl. GAA-containing fractions were concentrated using a Vivaspin 20 with a 10 kDa cutoff (Sartorius Stedim Biotech GmbH).
4
Purification of Lc-ALT Enzyme
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The crude LcALT was dialysed against 25 mM citrate buffer pH 6.2 and applied to a DEAE-Toyopearl-650 M (Tosoh Bioscience) column (22 × 90 mm) equilibrated with the same buffer (0.7 mL/min of flow rate). The fractions containing enzymatic activity were pooled and ammonium sulfate was added to achieve a final concentration of 400 mM. Subsequently, the protein solution was loaded onto a Phenyl-Toyopearl-650 M (Tosoh Bioscience) column (22 × 90 mm) equilibrated with 25 mM citrate buffer pH 6.2, containing 400 mM ammonium sulfate. The column was eluted with a 350–150 mM ammonium sulfate gradient with flow rate of 0.7 mL/min. Fractions exhibiting enzymatic activity were pooled, dialysed, and analysed on 8% SDS-PAGE gel.
5
Synthesis of Cobalamin Conjugates
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Chemicals: CN-Cbl, 1,1′-carbonyldiimidazole, and dodecylamine were purchased from Wako Pure Chemical Industries (Osaka, Japan). Phenyl-Toyopearl 650 M was purchased from Tosoh Corporation (Tokyo, Japan).
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