We synthesized N-terminal His-tagged YGFP derivatives, GFPuv and EGFP by standard PCR techniques and cloned them into the pEGFP vector at the Hind III and EcoR I sites, as described above. Protein expression was analyzed in DH5α cells at 37°C without any induction. After overnight incubation, cells were resuspended in PBS containing cOmplete™ Mini EDTA-free protease-inhibitor cocktail (Roche, Basel, Switzerland) and sonicated for 3 min (30 sec x 6 pulses) on ice with Q500 (Qsonica, LLC. Newton, CT, US). The soluble protein fraction was mixed with TALON Superflow Metal Affinity Resin (Takara Bio Inc) and incubated overnight at 4°C. After incubation, the resin was washed 5 times with PBS and then washed in PBS containing 200 mM imidazole to elute His-tagged proteins. Eluted proteins were subjected to VIVASPIN4 (Sartorius Stedim, Göttingen, Germany) centrifugal concentration for buffer exchange. To determine protein concentration, purified proteins dissolved in PBS were diluted 2-fold with 10% SDS and then incubated at 95°C for 10 min. Protein concentration of the quenched sample was determined using a BCA protein assay kit (Pierce Biotechnology, Rockland, IL, USA).
Talon superflow metal affinity resin
Manufactured by Takara Bio
Sourced in United States
TALON Superflow Metal Affinity Resin is a pre-packed chromatography resin designed for the purification of recombinant proteins. The resin utilizes a metal chelate ligand to selectively bind and capture target proteins. The high-flow-rate characteristics of the resin allow for efficient processing of large sample volumes.
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Lab products found in correlation
Amicon ultra 15 centrifugal filter unit, by Merck Group (2 mentions)
Vivaspin 4, by Sartorius (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Sepharose cl 4b, by Merck Group (1 mentions)
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
Celltracker green cmfda dye, by Thermo Fisher Scientific (1 mentions)
Celltracker deep red dye, by Thermo Fisher Scientific (1 mentions)
Celltracker red cmtpx dye, by Thermo Fisher Scientific (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Phospholipid, by Avanti Polar Lipids (1 mentions)
Cell culture flask, by Greiner (1 mentions)
Egg phosphatidylcholine, by Lipoid (1 mentions)
Jupiter c5, by Phenomenex (1 mentions)
Ultracel 100 membrane, by Merck Group (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Suz12, by Cell Signaling Technology (1 mentions)
Gelatin coated, by Merck Group (1 mentions)
Nanog, by Fortis Life Sciences (1 mentions)
12 protocols using talon superflow metal affinity resin
1
Purification of His-tagged Fluorescent Proteins
2
Large-Scale Production and Purification of MelBSt
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Cell growth for the large-scale production of WT MelBSt was carried out using the same expression vector and cell strain as used in the functional analyses (7 (link), 30 (link)). Briefly, MelBSt purification from membranes by cobalt-affinity chromatography (Talon Superflow Metal Affinity Resin, Takara) after extraction by 1.5% UDM. MelBSt protein was eluted with 250 mM imidazole in a buffer containing 50 mM NaPi, pH 7.5, 200 mM NaCl, 0.035% UDM, and 10% glycerol, and further dialyzed to change the buffer conditions accordingly.
3
Recombinant HEL-OVA Protein Production
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The nucleic acid sequence of HELWT, HEL2X (HEL with D101R and R73E mutations (Brink et al, 2008 (link))) or HEL3X (HEL with D101R, R73E, and R21Q mutations (Brink et al, 2008 (link))) fused to OVA217‐345 and a deka‐HIS tag was cloned into the pcDNA3.1 plasmid. Expi293 or HEK293E cells were transfected using polyethyleneimine (PEI) following culture for 5 days. TALON Superflow Metal Affinity Resin (Takarabio cat. 635506) was used to purify the recombinant HEL‐OVApep proteins. After dialysis against PBS, and concentration to 0.8–1.2 mg/ml (Amicon Ultra‐15 Centrifugal Filter Units, Merck cat. UFC901024), the final proteins were analyzed by polyacrylamide gel electrophoresis and Coomassie blue staining, aliquoted and frozen at −80°C.
4
Large-Scale Production and Purification of MelBSt Protein
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Cell growth for the large-scale production of WT MelBSt was carried out using the pK95 ΔAH/MelBSt/CHis10 from E. coli DW2 cells (melA+melB, lacZY) as described (10 (link), 39 (link)). Briefly, MelBSt purification from membranes by cobalt-affinity chromatography (Talon Superflow Metal Affinity Resin, Takara) after extraction by 1.5% UDM or 2% DDM. MelBSt protein was eluted with 250 mM imidazole in a buffer containing 50 mM NaPi, pH 7.5, 200 mM NaCl, 0.035% UDM or 0.01% DDM, and 10% glycerol, and dialyzed to change the buffer conditions accordingly.
5
Recombinant HEL-OVApep Protein Purification
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The nucleic acid sequence of HEL WT , HEL 2X (HEL with D101R and R73E mutations [73] ) or HEL 3X (HEL with D101R, R73E and R21Q mutations [73] ) fused to OVA217-345 and a deka-HIS tag was cloned into the pcDNA3.1 plasmid. Expi293 or HEK293E cells were transfected using polyethyleneimine (PEI) following culture for 5 days. TALON Superflow Metal Affinity Resin (Takarabio cat. 635506) was used to purify the recombinant HEL-OVApep protein. After dialysis against PBS, and concentration to 0.8-1.2 mg/mL (Amicon Ultra-15 Centrifugal Filter Units, Merck cat. UFC901024), the final protein was analyzed by polyacrylamide gel electrophoresis and Coomassie blue staining, aliquoted and frozen at -80°C.
6
Purification of Recombinant Proteins from E. coli
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E. coli BL21 (λDE3) cells harboring recombinant plasmids were grown at 37 °C in LB medium supplemented with 100 μg/mL ampicillin until the culture reached mid log phase (0.3–0.4, OD600). Isopropyl-1-thio-β-d -galactoside (IPTG) was added to the culture to the final concentration of 0.5 mM. The culture was incubated at 20 °C and shaken for overnight. The bacterial cells were harvested and protein purification was carried out by Immobilized Metal ion Affinity Column (IMAC) method using Talon SuperFlow Metal Affinity Resin (Clontech, Mountain View, CA, USA) as recommended by the manufacturer. The protein was dialyzed (to remove immidazole) and concentrated using 3 kDa Amicon Ultra centrifugal filter (Millipore, Burlington, MA, USA), and kept in 1× equilibrium buffer (50 mM Sodium phosphate, 300 mM NaCl, pH 7.0) and 50% glycerol. The enzyme was expressed in a good performance with an expected size of 74 kDa on SDS-PAGE.
7
Recombinant M-CTX-Fc Protein Production
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The preparation of M-CTX-Fc was performed as previously described [29 ]. Escherichia coli BL21 (DE3) pLysS (Novagen) was transformed with the expression vector for M-CTX-Fc. After induction of the expression vector, the transformant was cultured and the bacteria were harvested. The inclusion bodies were washed and then were dissolved in 6 M guanidinium-HCl containing 0.1 M Tris-HCl (pH 8.5). The protein in the solution was reduced and then refolded. The solution containing refolded protein was purified using a cobalt resin column (Talon Superflow Metal Affinity Resin, Clontech, Mountain View, CA, USA). The eluted solution was dialyzed thrice using phosphate-buffered saline (Dulbecco's formula, hereafter PBS). The purity of M-CTX-Fc in the final preparation was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie Brilliant Blue (CBB) staining, and western blotting.
8
Nanobody-based Protein Labeling Protocol
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MicroBCA Protein Assay Kit, AlexaFluor 488 NHS ester, CellTracker Green CMFDA dye, CellTracker Deep Red dye, CellTracker Red CMTPX dye and the pcDNA3.1 vector with Neomycin resistance were purchased from Thermo Fisher Scientific (Waltham, USA). Sepharose CL-4B and cholesterol were obtained from Sigma-Aldrich (Steinheim, Germany) and TALON Superflow Metal Affinity Resin was from Clontech Laboratories, Inc. (Saint-Germain-en-Laye, France). Phospholipids were purchased from Avanti Polar Lipids (Birmingham, USA), except for egg phosphatidylcholine, which was obtained from Lipoid AG (Steinhausen, Switzerland). Cell culture flasks were obtained from Greiner Bio-One (Alphen aan de Rijn, The Netherlands). pET28a-EGa1 and pAX51-R2 vectors encoding EGa1 (PDB ID: 4KRN) and R2 (PDB ID: ; 1QD0) Myc-tagged nanobodies, respectively, were kindly provided by Dr S. Oliveira (Department of Biology, Utrecht University, Utrecht, The Netherlands).
9
Purification of Recombinant Polypeptides APHC1-3
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Polypeptides APHC1-3 were produced as previously described [24 (link)]. DNA fragments encoding the polypeptides were cloned into the expression vector pET32b+ (Novagen, USA). Escherichia coli BL21 (DE3) cells expressing thioredoxin fusions of polypeptides were cultured overnight at 25°C, harvested, ultrasonicated in a buffer for metal-affinity chromatography, and centrifuged to remove all insoluble particles. Fusion proteins were purified using a TALON Superflow Metal Affinity Resin (Clontech, USA) and subjected to CNBr cleavage, as described [30 (link)]. The recombinant polypeptides were purified on a reverse-phase column Jupiter C5 (Phenomenex, USA) 250×10 mm. The target polypeptides’ purity was verified by MALDI-TOF mass-spectrometry.
10
Membrane Protein Purification Protocol
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