A HaloTag-fused Streptococcus pyogenes Cas9 protein containing the double nuclease mutation (D10A and H840A; dCas9-Halo) was expressed in Escherichia coli strain BL21 (DE3) using the pET302-6His-dCas9-Halo plasmid (Deng et al., 2015 ; Addgene plasmid no. 72269). After induction with 1 mM isopropyl β-d -1-thiogalactopyranoside, the 6His-dCas9-Halo proteins were purified using TALON Single Step Columns (Clontech, Palo Alto, CA, USA) following the manufacturer’s instructions. The purified proteins were concentrated using Amicon Ultra 100 K filters (Merck Millipore, Burlington, MA, USA) and were exchanged into a storage buffer [50 mM HEPES (pH 7.5), 150 mM KCl, 1 mM Tris(2-carboxyethyl)phosphine, 20% (v/v) glycerol]. The dCas9 proteins were stored at –20 °C.
Talon single step columns
Manufactured by Takara Bio
Sourced in United States
The TALON Single Step Columns are affinity chromatography columns designed for the purification of recombinant proteins. They utilize a cobalt-based IMAC resin to efficiently capture and purify histidine-tagged proteins from cell lysates or other sample types.
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2 protocols using talon single step columns
1
Purification of Cas9-HaloTag Fusion Protein
2
Purification of Recombinant HaRVT Proteins
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Lysates were prepared in lysis buffer (50 mM phosphate buffer, 300 mM NaCl, pH 7.0) for metal-affinity chromatography, or in buffer A (50 mM Tris-HCl, 100 mM NaCl, 1:1000 2-Mercaptoethanol, pH 7.5) for sucrose gradient separation; both were supplemented with Roche cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma) according to manufacturer's instructions.
Frozen cell pellets containing induced protein were thawed in the respective buffer and sonicated on ice (10 s followed by 30-s pause for 3 times). Soluble proteins were separated from insoluble debris by centrifugation at 4ºC, 4000 g for 30 min.
Recombinant proteins were purified using Co 2+ -based immobilized metal affinity chromatography (IMAC). We used HisTALON™ Gravity Column Purification Kit (Clontech) for HaRVT and TALON® Single Step Columns (Clontech) for other HaRVT mutant versions, following the manufacturers' protocols. Roche cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma) was added to all buffers. Purified proteins were stored at 4ºC.
Frozen cell pellets containing induced protein were thawed in the respective buffer and sonicated on ice (10 s followed by 30-s pause for 3 times). Soluble proteins were separated from insoluble debris by centrifugation at 4ºC, 4000 g for 30 min.
Recombinant proteins were purified using Co 2+ -based immobilized metal affinity chromatography (IMAC). We used HisTALON™ Gravity Column Purification Kit (Clontech) for HaRVT and TALON® Single Step Columns (Clontech) for other HaRVT mutant versions, following the manufacturers' protocols. Roche cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma) was added to all buffers. Purified proteins were stored at 4ºC.
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