The purification protocol for both protein constructs was performed by immobilised-metal affinity chromatography (IMAC) using a HisTrap™ column (5 mL) with Ni2+, coupled to the chromatograph ÄKTA START (GE Healthcare). The cells pellet was resuspended in their respective Start Buffer (Table 1 ) containing protease inhibitors, DNaseI (5 µg/mL), 5 mM MgCl2 and Lysozyme (300 µg/mL). Cells were disrupted by sonication (UP100H, Hielscher Ultrasonics) and the crude extract centrifuged at 13,000×g for 30 min (Eppendorf, Centrifuge 5804K). The supernatant was loaded into the column, and the first wash with 50 mM imidazole was applied. Protein was eluted using an imidazole gradient from 50 to 500 mM. All fractions were analysed by SDS-PAGE and pooled together. Proteins were buffer-exchanged to an appropriate storage buffer (Table 1 ) with no imidazole, using HiTrap Desalting columns with Sephadex G-25 resin (GE Healthcare), coupled to the ÄKTA START. Purified proteins were concentrated by ultrafiltration using Vivaspin® Turbo 15 (Sartorius) with a 3 kDa cut-off membrane.
Kta start
Manufactured by GE Healthcare
Sourced in United States, Sweden
The ÄKTA Start is a compact and easy-to-use liquid chromatography system designed for protein purification. It is capable of performing a range of chromatography techniques, including affinity, ion exchange, and size exclusion chromatography. The system is controlled by an intuitive software interface and features automated sample injection, fraction collection, and buffer selection.
Automatically generated - may contain errors
Lab products found in correlation
Hrv3c processing tag, by GenScript (3 mentions)
Avitag, by GenScript (3 mentions)
Freestyle 293f expression system, by Thermo Fisher Scientific (3 mentions)
Histidine tag, by GenScript (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hisprep ff 16 10, by GE Healthcare (1 mentions)
Vivaspin 20, by GE Healthcare (1 mentions)
T s series machine, by Constant Systems (1 mentions)
Protease inhibitor cocktail, by Abcam (1 mentions)
Hiload superdex 75pg, by GE Healthcare (1 mentions)
Tricorn 5 50 column, by GE Healthcare (1 mentions)
Optima max xp, by Beckman Coulter (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Unicorn software, by GE Healthcare (1 mentions)
5804r centrifuge, by Eppendorf (1 mentions)
Superdex 75 10 300 gl, by GE Healthcare (1 mentions)
Vivaspin concentrator, by Sartorius (1 mentions)
Hiprep 16 60 sephacryl s 200 hr, by GE Healthcare (1 mentions)
Penicillin g streptomycin, by Thermo Fisher Scientific (1 mentions)
Kta pure, by GE Healthcare (1 mentions)
21 protocols using kta start
1
Purification of Protein Constructs by IMAC
2
Recombinant PfCSP Expression and Purification
3
Production and Purification of VHH Antibodies
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Selection and characterization of VHH clones targeting Met have been described previously [25 (link)]. VHH protein for epitope mapping was obtained from production in E. coli TG1. For this, the VHH genes were cloned into the pMEK222 vector for productions in E. coli, which provides the VHH with a C-terminal FLAG-His tag. VHHs were produced and purified from E. coli TG1 using immobilized metal-affinity chromatography (IMAC, Thermo Fisher Scientific, Waltham, MA, USA) as previously described [7 (link),28 (link)]. For production in yeast, VHH genes were recloned in the pYQVQ11 vector for VHH production in yeast, which provides the VHH with a C-terminal C-Direct tag containing a free thiol (cysteine) and an EPEA (Glu, Pro, Glu, Ala) purification tag (C-tag, Thermo Fisher Scientific). To improve production yields and facilitate purification from supernatant, C-Direct-tagged VHH were produced in S. cerevisiae strain VWK18 as described previously [28 (link),29 (link),30 (link),31 (link)]. VHHs were purified from the yeast supernatant using an Äkta Start (GE Healthcare, Chicago, IL, USA), a Capture-Select affinity chromatography column (Thermo Fisher Scientific) and size-exclusion chromatography (Thermo Fisher Scientific) according to the manufacturer’s protocols. Purified VHH was filter sterilized and stored in PBS (phosphate buffered saline).
4
AKTA Start Liquid Chromatography
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All subsequent chromatography steps were performed with an ÄKTA Start liquid chromatography system (GE Healthcare).
5
Recombinant Protein Purification Protocol
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Cell lysis was done with a high‐pressure cell disrupter (T‐S Series Machine, Constant Systems Limited). Following centrifugation, the supernatant was purified by Ni‐immobilized metal affinity column (IMAC), (Äkta start, GE Healthcare, USA or manual). After loading the supernatant on a HisPrep FF 16/10 or manual packed column (GE Healthcare, USA), the column was washed with 4–5 column volumes (CV) of 20 mm Tris‐HCl followed by 4–5 CV of 2 mm imidazole in 20 mm Tris‐HCl, pH 8. The protein was eluted with 200 mm imidazole in 20 mm Tris‐HCl. After dialysis against 20 mm Tris‐HCl, pH 8, the protein was analyzed by SDS‐PAGE for quality control. Depending on the solubility of the construct, the proteins were concentrated to 200–400 mg mL−1 with centrifugal concentrators (Vivaspin 20, 10 kDa MWCO, GE Healthcare, USA) and then frozen at −20 °C until further use.
6
Purification of Calmodulin Variants
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CaM-WT and E140G variant were expressed and purified as previously described (79 (link)). In short, CaM pESUMOPro-Kan plasmids were transformed into Escherichia coli BL21(DE3) STAR and cultured in 2xYT media containing 100 μg/ml kanamycin. Expression was induced with 0.5 mM IPTG overnight at 18 °C. Cells were harvested by centrifugation and pellets were resuspended in 50 mM Hepes, 200 mM NaCl, pH 7.5 supplemented with protease inhibitor cocktail (Proteoloc, Abcam). Cells were lysed with lysozyme (1 mg/ml) for 30 min on ice followed by sonication. Lysates were further treated with BaseMuncher (Abcam) and clarified by ultracentrifugation.
Clarified lysates were purified on a HisTrap HP column (ÄKTA Start, GE Healthcare) using a linear gradient of 0 to 500 mM imidazole. Eluted proteins were dialyzed overnight at 4 °C (8 kDa) to remove the imidazole, and His-tag was removed by treatment with SUMO protease (ULP1). CaM proteins were then further purified by reverse HisTrap and size-exclusion chromatography (HiLoad Superdex 75pg, ÄKTA Pure, GE Healthcare). Fractions containing the purified proteins were concentrated using Amicon centrifugation units (3 kDa), flash-frozen in liquid nitrogen, and stored in -80 °C until used.
Clarified lysates were purified on a HisTrap HP column (ÄKTA Start, GE Healthcare) using a linear gradient of 0 to 500 mM imidazole. Eluted proteins were dialyzed overnight at 4 °C (8 kDa) to remove the imidazole, and His-tag was removed by treatment with SUMO protease (ULP1). CaM proteins were then further purified by reverse HisTrap and size-exclusion chromatography (HiLoad Superdex 75pg, ÄKTA Pure, GE Healthcare). Fractions containing the purified proteins were concentrated using Amicon centrifugation units (3 kDa), flash-frozen in liquid nitrogen, and stored in -80 °C until used.
7
Protein A Adsorption Isotherms and Dynamics
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IgG adsorption isotherms on the non‐grafted and CMD‐grafted protein A gels were determined in 20 mmol/L phosphate buffer at different pHs (pH 4.5, 7.4 or 10.0) and NaCl concentrations (0−150 mmol/L) as described by Yang et al. [18 ]. Saturated adsorption capacity (qm, mg/g gel) and dissociation constant (Kd, mg/mL) of IgG were obtained by fitting the experimental data to Langmuir model as follows,
Dynamic binding capacities (DBC) for IgG onto the non‐grafted and CMD‐grafted protein A gel were measured in a Tricorn 5/50 column connected to an ÄKTA Start (GE Healthcare, Uppsala, Sweden). In the experiment, 100 mmol/L NaCl in 20 mmol/L phosphate buffer (pH 10.0) was applied as binding buffer, and the preparation of IgG solution. After the column was equilibrated with binding buffer, 0.5 mg/mL IgG solution was loaded at 0.5 mL/min. After IgG breakthrough, the column was washed with binding buffer to remove free protein and the adsorbed IgG was eluted by 0.01 mol/L glycine‐HCl buffer (pH 3.0). Finally, the column was regenerated by 0.1 mol/L NaOH for next experiments. DBC for IgG was calculated as follows,
where V10 and VB are loading volume at 10% breakthrough and the column volume, respectively; V0 is the dead volume measured with 2% acetone aqueous solution.
Dynamic binding capacities (DBC) for IgG onto the non‐grafted and CMD‐grafted protein A gel were measured in a Tricorn 5/50 column connected to an ÄKTA Start (GE Healthcare, Uppsala, Sweden). In the experiment, 100 mmol/L NaCl in 20 mmol/L phosphate buffer (pH 10.0) was applied as binding buffer, and the preparation of IgG solution. After the column was equilibrated with binding buffer, 0.5 mg/mL IgG solution was loaded at 0.5 mL/min. After IgG breakthrough, the column was washed with binding buffer to remove free protein and the adsorbed IgG was eluted by 0.01 mol/L glycine‐HCl buffer (pH 3.0). Finally, the column was regenerated by 0.1 mol/L NaOH for next experiments. DBC for IgG was calculated as follows,
where V10 and VB are loading volume at 10% breakthrough and the column volume, respectively; V0 is the dead volume measured with 2% acetone aqueous solution.
8
Ultracentrifugation-based Lipid Particle Separation
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Ultracentrifugation
allows samples
to be separated by mass and density upon application of a centrifugal
force. Samples for ultracentrifugation experiments were prepared at
a ratio of 1:9 labeled/unlabeled with the final lipid concentration
of labeled particles being 267 μM. The samples were supplemented
with 10 mM CaCl2 and incubated for 30 min. Calcium was
subsequently chelated using 5 equiv of EDTA and 2 equiv of EGTA. For
loading in the ultracentrifugation tube, the samples were prepared
by mixing 1:1 with 70% sucrose solution using a 20 mM Tris 100 mM
NaCl 0.5 mM EDTA pH 7.4 buffered solution and placing 400 μL
of the resulting 35% gradient layer in the bottom of an open-top thickwall
polycarbonate tube (3.5 mL, 13 × 51 mm; Beckman Coulter). Following
this, 400 μL layers of buffer 30, 25, 20, 15, 10% sucrose solutions
were carefully layered in 200 μL increments to minimize mixing.
The final layer consisted of ∼400 μL of buffer with volume
adjusted as needed to balance the centrifuge tubes. The samples were
then spun for 3 h at 268,000g in a precooled (4 °C)
Optima MAX-XP (Beckman Coulter) centrifuge. After the centrifugation,
fractions were collected from the bottom of the tube using a long
needle connected to an ÄKTA Start (GE Healthcare) chromatographic
system.
allows samples
to be separated by mass and density upon application of a centrifugal
force. Samples for ultracentrifugation experiments were prepared at
a ratio of 1:9 labeled/unlabeled with the final lipid concentration
of labeled particles being 267 μM. The samples were supplemented
with 10 mM CaCl2 and incubated for 30 min. Calcium was
subsequently chelated using 5 equiv of EDTA and 2 equiv of EGTA. For
loading in the ultracentrifugation tube, the samples were prepared
by mixing 1:1 with 70% sucrose solution using a 20 mM Tris 100 mM
NaCl 0.5 mM EDTA pH 7.4 buffered solution and placing 400 μL
of the resulting 35% gradient layer in the bottom of an open-top thickwall
polycarbonate tube (3.5 mL, 13 × 51 mm; Beckman Coulter). Following
this, 400 μL layers of buffer 30, 25, 20, 15, 10% sucrose solutions
were carefully layered in 200 μL increments to minimize mixing.
The final layer consisted of ∼400 μL of buffer with volume
adjusted as needed to balance the centrifuge tubes. The samples were
then spun for 3 h at 268,000g in a precooled (4 °C)
Optima MAX-XP (Beckman Coulter) centrifuge. After the centrifugation,
fractions were collected from the bottom of the tube using a long
needle connected to an ÄKTA Start (GE Healthcare) chromatographic
system.
9
DNA Template Purification by FPLC and Desalting
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Chromatographic purification was performed on the ÄKTA Start (GE Healthcare) FPLC system composed of two pumps and a multiwavelength UV-Vis detector (2 mm flow cell path length). Unicorn software (GE Healthcare) was used for instrument control and data acquisition. DNA template linearized was diluted once in sample loading buffer (75 mM Tris + 15 mM EDTA + 3.75 M SA (ammonium sulphate) pH = 7.2 and loaded onto CIMmultus C4 HDL 1 mL column (Sartorius) equilibrated in mobile phase containing 50 mM Tris + 10 mM EDTA + 2.5 M SA pH = 7.2. After the UV 260 nm signal and conductivity was stabilized, an elution step was performed with 50 mM Tris + 10 mM EDTA pH = 7.2 DNA content from desired fractions was concentrated and desalted using Amicon Ultra-15 centrifugal filter units (30K membrane) (Millipore) by successive centrifugation at 1,000 g for 10 min RT in a 5804R centrifuge (Eppendorf).
10
Recombinant PfCSP Expression and Purification
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Recombinant PfCSP constructs were produced as previously described.8 (link) Briefly, sequences corresponding to rPfCSPFL and Junctional region (rPfCSP5/3) were cloned into the same CMV/R-expression vectors with a C-terminal AviTag, HRV3C-processing tag, and a 6X histidine tag (GenScript). rPfCSP constructs were expressed through transient transfection in 293F cells using the Freestyle 293F expression system (Thermo Fisher Scientific) at 37°C, 8% CO2 for 6 days, and purified from culture supernatants through polyhistidine-tag affinity chromatography followed by size exclusion chromatography (SEC) on an ÄKTA™ Start (GE Healthcare). Monomer-containing fractions were pooled, concentrated, snap frozen, and stored at −80°C. Peptides 21, 22, and 29 were produced by direct synthesis and biotinylated by GenScript.
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