E. coli Rosetta (DE3) cells containing pRARE plasmids encoding tRNAs for rare codons were transformed with either WT plasmid pET21c-PheRS (a gift from L. Spremulli, University of North Carolina, Chapel Hill, NC, USA) or mutant mtPheRS plasmids P85A and H135D. Point mutations were introduced by site directed mutagenesis using the QuickChange protocol (Stratagene). Cultures were grown at 37°C to OD = 0.8 and induced with 0.5 mM IPTG for 4 h. Cells were harvested, sonicated and supernatant was collected after centrifugation at 25 000×g for 1 h. The supernatant was applied to a TALON metal affinity resin column (Clontech), followed by washing and the protein was eluted with 25 mM Tris–HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole and 10% glycerol. Fractions containing mtPheRS were checked for electrophoretic purity by SDS–PAGE, pooled and dialyzed overnight at 4°C in 50 mM Tris–HCl, pH 7.5, 5 mM MgCl2 and 100 mM KCl. The purified enzyme was concentrated, adjusted to 50% (v/v) glycerol and aliquots were stored at -80°C. E. coli tRNAPhe was made by T7 runoff transcription as previously described13 (link); 10 (link). DNA template for tRNA transcription was generated from plasmids carrying tRNA genes by PCR amplification. The tRNA was phenol chloroform extracted and purified on a DEAE sepharose column, as described previously13 (link), 10 (link).
Talon metal affinity resin column
Manufactured by Takara Bio
Sourced in France
The TALON metal affinity resin column is a chromatography resin designed for the purification of histidine-tagged recombinant proteins. The resin contains chelated cobalt ions that bind to the histidine tags, allowing the target proteins to be captured and separated from other cellular components.
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Lab products found in correlation
4 protocols using talon metal affinity resin column
1
Purification of Mutant Phenylalanyl-tRNA Synthetase
2
Purification of αSyn and HTTExon1 Proteins
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Recombinant human wild-type αSyn was expressed in E. coli strain BL21(DE3) (Stratagene, La Jolla, CA, USA) and purified as described43 (link). Recombinant C-terminally hexa His-tagged MBP-TEV-HTTExon1-His with a polyQ stretch of 48 glutamine residues was expressed in E. coli strain BL21(DE3) (Stratagene, La Jolla, CA, USA) and purified as described44 (link). HTTExon1-His was obtained from MBP-TEV-HTTExon1-His as described44 (link). Briefly, MBP-TEV protease was incubated with the purified MBP-TEV-HTTExon1-His until 100% cleavage was achieved. HTTExon1-His was purified from the resulting mixture using a Talon metal affinity resin column (Clontech, Saint-Germain-en-Laye, France), immediately filtered through a 0.22 μM filter, aliquoted, flash frozen in liquid nitrogen and stored at −80 °C until use.
3
Recombinant TAT Fusion Protein Purification
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TAT-HA-SNX9 and TAT-HA-Smad3 proteins were prepared in BL21(DE3)pLysS Escherichia coli (OD600 of 0.4) following addition of isopropyl β-d -thiogalactopyranoside to a final concentration of 0.5 mM. After a 4-h TAT protein induction at 37°C, cells were harvested, washed, and suspended in 15 ml 50 mM sodium phosphate buffer (pH 7.4) containing 300 mM NaCl and 20 mM imidazole. The cell suspension was sonicated, and the lysate was cleared by 12,000 × g centrifugation at 4°C for 20 min. The supernatant was then poured into a TALON Metal Affinity Resin column (Clontech, Mountain View, CA), washed with 50 mM sodium phosphate buffer containing 40 mM imidazole, and eluted with 150 mM imidazole.
4
Purification of EngBF Mutant Variants
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EngBF mutant variants were constructed by the two-reaction site directed mutagenesis procedure as described previously 18 using the following oligo-deoxyribonucleotides, H718Q EngBF and mutant variants as well as the sialidase, NanI, used for asialo-CGMP preparation were produced, purified and their concentration estimated as previously described 18 . Larger preparations of EngBF and mutant variants to be used with rapid quench flow experiments were purified from 0.1 to 0.5 L cultures where the supernatant from the sonicate in binding buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4) was loaded on to a 10 mL Talon Metal Affinity Resin column (Clontech), washed with the binding buffer and eluted with a gradient from 0 to 500 mM imidazole in the same buffer. Fractions containing purified protein were pooled and dialyzed against 50 mM sodium acetate, 50 mM NaCl, pH 4.5 and stored at 4 °C.
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