His tagged RBD was purified from clarified supernatants via a batch bind method where each clarified supernatant was supplemented with 1 M Tris-HCl pH 8.0 to a final concentration of 45 mM and 5 M NaCl to a final concentration of 310 mM. Talon cobalt affinity resin (Takara) was added to the treated supernatants and allowed to incubate for 15 min with gentle shaking. Resin was collected using vacuum filtration with a 0.2 mm filter and transferred to a gravity column. The resin was washed with 20 mM Tris pH 8.0, 300 mM NaCl, and the protein was eluted with 3 column volumes of 20 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole. The batch bind process was then repeated and the first and second elutions combined. SDS-PAGE was used to assess purity. Following IMAC purification, the elution was concentrated and applied to a Cytiva S200 Increase column equilibrated with 20mM Tris 150mM NaCl pH8.0, and the peak of interest was collected and quantified using A280. The purified RBD was qualified using BLI to confirm binding using CR3022 and hACE2-Fc.
Talon cobalt affinity resin
Manufactured by Takara Bio
Sourced in Japan
TALON cobalt affinity resin is a chromatography resin designed for the purification of recombinant proteins. It utilizes a cobalt-based metal affinity ligand to selectively bind and capture proteins with histidine-tags. The resin offers high binding capacity and selectivity, providing efficient purification of target proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay kit, by Bio-Rad (3 mentions)
Sephadex g 25m column, by GE Healthcare (3 mentions)
10k molecular weight cutoff dialysis cassette, by Thermo Fisher Scientific (2 mentions)
Bugbuster reagent, by Merck Group (1 mentions)
N decyl β d maltopyranoside, by Anatrace (1 mentions)
Superdex 200 10 30 gl size exclusion column, by GE Healthcare (1 mentions)
Ni nta chromatography, by GoldBio (1 mentions)
Carboxypeptidase a, by Merck Group (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Sonifier 450, by Emerson (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
Myokinase, by Merck Group (1 mentions)
Slide a lyzer mini dialysis device, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
14 protocols using talon cobalt affinity resin
1
Purification and Characterization of SARS-CoV-2 RBD
2
SARS-CoV-2 RBD Purification and Characterization
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His-tagged RBD was purified from clarified supernatants via a batch bind method, where each clarified supernatant was supplemented with 1 M Tris-HCl, pH 8.0, to a final concentration of 45 mM and 5 M NaCl to a final concentration of 310 mM. Talon cobalt affinity resin (Takara Bio) was added to the treated supernatants and allowed to incubate for 15 min with gentle shaking. Resin was collected using vacuum filtration with a 0.2-mm filter and transferred to a gravity column. The resin was washed with 20 mM Tris, pH 8.0, 300 mM NaCl, and the protein was eluted with 3 CVs of 20 mM Tris, pH 8.0, 300 mM NaCl and 300 mM imidazole. The batch bind process was then repeated and the first and second elutions combined. SDS-PAGE was used to assess purity. Following immobilized metal affinity chromatography purification, the elution was concentrated and applied to a Cytiva S200 Increase column equilibrated with 20 mM Tris 150 mM NaCl, pH 8.0, and the peak of interest was collected and quantified using A280. The purified RBD was qualified using BLI to confirm binding using CR3022 and hACE2-Fc.
3
Purification of SARS-CoV-2 Spike Proteins
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Proteins were purified from clarified supernatants via a batch bind method where each supernatant was supplemented with 1 M Tris-HCl pH 8.0 to a final concentration of 45 mM and 5 M NaCl to a final concentration of ~310 mM). Talon cobalt affinity resin (Takara) was added to the treated supernatants and allowed to incubate for 15 minutes with gentle shaking. Resin was collected using vacuum filtration using a 0.2 μm filter and transferred to a gravity column. The resin was washed with 20 mM Tris pH 8.0, 300 mM NaCl, and the protein was eluted with three column volumes of 20 mM Tris pH 8.0, 300 mM imidazole, 300 mM NaCl. The batch bind process was then repeated and the first and second elutions combined. SDS-PAGE was used to assess purity. Purified S-2P trimer was concentrated to ~1 mg/mL and dialyzed into 50 mM Tris pH 8, 150 mM NaCl, 0.25% L-Histidine, 5% glycerol in a hydrated 10k molecular weight cutoff dialysis cassette (Thermo Scientific). The purified RBD protein was dialyzed into 50 mM Tris pH 7, 185 mM NaCl, 100 mM Arginine, 4.5% glycerol, 0.75% w/v CHAPS. Due to inherent instability, S-2P was immediately flash frozen and stored at −80°C.
4
Purification of SARS-CoV-2 S-2P and RBD Proteins
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Proteins were purified from clarified supernatants via a batch bind method where each supernatant was supplemented with 1 M Tris-HCl pH 8.0 to a final concentration of 45 mM and 5 M NaCl to a final concentration of ∼310 mM). Talon cobalt affinity resin (Takara) was added to the treated supernatants and allowed to incubate for 15 min with gentle shaking. Resin was collected using vacuum filtration using a 0.2 μm filter and transferred to a gravity column. The resin was washed with 20 mM Tris pH 8.0, 300 mM NaCl, and the protein was eluted with three column volumes of 20 mM Tris pH 8.0, 300 mM imidazole, 300 mM NaCl. The batch bind process was then repeated and the first and second elutions combined. SDS-PAGE was used to assess purity. Purified S-2P trimer was concentrated to ∼1 mg/mL and dialyzed into 50 mM Tris pH 8, 150 mM NaCl, 0.25% L-histidine, 5% glycerol in a hydrated 10k molecular weight cutoff dialysis cassette (Thermo Scientific). The purified RBD protein was dialyzed into 50 mM Tris pH 7, 185 mM NaCl, 100 mM arginine, 4.5% glycerol, 0.75% w/v CHAPS. Due to inherent instability, S-2P was immediately flash frozen and stored at −80°C.
5
Purification of Recombinant KAI2 Proteins
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AtKAI2 and BtKAI2-coding sequences were cloned into pE-SUMO-Amp (LifeSensors) to generate N-terminal 6 × HIS-SUMO fusion proteins. All proteins were expressed in BL21 Rosetta DE3 pLysS cells (Merck-Millipore) and purified using IMAC as described in detail previously41 (link). In brief, cultures were grown in Luria-Bertani medium at 30 °C until the optical density reached 0.8–1, at which point the cultures were chilled to 16 °C and induced with 0.1 mM isopropyl β-d -1 thiogalactopyranoside and allowed to grow for a further 14–16 h. Pellets were lysed in 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 10 mM imidazole and 1 × BugBuster reagent (Merck-Millipore). Proteins were purified from lysates in batch mode using gravity columns containing TALON cobalt affinity resin (Takara). Columns were washed with 10 mM imidazole and eluted with 200 mM imidazole in 1-mL fractions. Proteins were concentrated and imidazole removed by ultrafiltration and buffer exchange into 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol.
6
Purification and Labeling of CLC-ec1 Mutants
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DNA constructs for CLC-ec1 C85A/H234C (WT), and C85A/H234C/R230C/L249C (RCLC) have been described earlier, along with their expression and purification methods (4 (link), 6 (link)). Briefly, BL21-AI E. coli cells transformed with the expression plasmid were lysed by sonication and the protein extracted from membrane fragments into 2% n-decyl-β-D-maltopyranoside (DM; Anatrace) containing 5 mM TCEP [Tris (2-carboxyethyl) phosphine; Soltec Bioscience] to ensure that the introduced cysteine residue at residue 234 (H234C) remains in a reduced state for maleimide labeling. After pelleting cellular debris by centrifugation, the protein was affinity purified using TALON cobalt affinity resin (Clontech Laboratories) followed by size exclusion chromatography on a Superdex 200 10/30 GL size exclusion column (GE Healthcare) into size exclusion buffer (SEB): 150 mmol L-1 (mM) NaCl, 20 mM MOPS pH 7.0, and 5 mM analytical-grade DM. Molar extinction coefficients for WT and RCLC are 46,020 M−1 cm−1 and 49,630 M−1 cm−1 respectively, and molecular weights for WT and RCLC are 52,000 g/mol and 49,630 g/mol respectively.
7
Nanobody Expression and Purification
8
Recombinant Protein Production Protocols
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Recombinant HA-tagged Gln4 protein for use as a standard was produced by transforming E. coli strain Rosetta-gami B (DE3; Novagen) with pET19b-GLN4. Protein expression was induced in pET19b-GLN4 transformants using 1 mM IPTG for 3 h. Following harvest and lysis by sonication, TALON cobalt affinity resin (Clontech) was used to purify the His-tagged protein using manufacturer's protocols, Samples were dialysed to remove excess salt and imidazole before mass spectrometry analysis using a Q-Exactive Mass Spectrometer (ThermoFisher) to confirm protein identity. Recombinant GFP for use as a standard for the determination of ribosome content was similarly produced using pET19b-GFP.
9
Expression and Purification of FACL6 Protein
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Expression of FACL6 in pET200 D-TOPO construct was induced with IPTG in a 600 ml culture of E. coli BL21 Star (DE3). The cells were washed and resuspended in lysis buffer (50 mM sodium phosphate pH 7.4, 300 mM NaCl with a cocktail of protease inhibitors) and were disrupted by sonication using a Branson Sonifier 450 (Branson Ultrasonics Corp.). The cell lysates were clarified by centrifugation at 16,000 x g, 4°C and the pellet was used for purification of the expressed protein since our attempts to purify the protein from supernatant were unsuccessful. The pellet was solubilized in 5 ml ice-cold lysis buffer with 1% (w/v) dodecylmaltoside and 0.03% Triton X-100 for 15 min with intermittent sonication to enhance solubilization of the inclusion bodies. The solution was centrifuged and the supernatant was used to purify the FACL6 protein using a 5-ml bed volume TALON cobalt-affinity resin (Clontech, CA) according to the manufacturer’s protocol. The column was washed with 10 mM imidazole and bound protein was eluted with 200 mM imidazole. The proteins in each fraction were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by coomassie staining.
10
Purification of His-tagged Proteins
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