The anti-E2 murine MAbs AP33 and ALP98, and the rat MAb 3/11 have been described previously57 (link)–59 (link). The anti-idiotype murine MAb B2.1 A was generated as described herein. MAbs were purified from hybridoma supernatant on HiTrap protein G columns using an Äkta Purifier (GE Healthcare). Fab fragments of AP33 and B2.1 A were made by digesting the respective IgGs for 7 h with immobilized papain, followed by purification using a Nab protein A Plus column (Thermofisher). AP33 Fab was further purified for crystallization by anion exchange on Mono Q 5/50 GL (GE Healthcare) in 20 mM Tris pH 8.5, using a gradient of 0–300 mM NaCl. Biotinylation was carried out using the ImmunoprobeTM biotinylation kit (Sigma, BK101). B2.1 A Fab was purified for surface plasmon resonance (SPR) by size exclusion chromatography (SEC) on Superdex 200 GL (GE Healthcare).
Kta purifier
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Germany, Sweden, Spain
The ÄKTA purifier is a versatile chromatography system designed for protein purification. It is capable of performing various chromatographic techniques, including affinity, ion exchange, and size exclusion, to separate and purify target proteins from complex mixtures. The system features automated control, monitoring, and data collection capabilities to ensure reliable and reproducible results.
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Lab products found in correlation
Superdex 200 10 300 gl column, by GE Healthcare (10 mentions)
Astra 6, by Wyatt Technology (7 mentions)
Hitrap heparin column, by GE Healthcare (5 mentions)
Hitrap q column, by GE Healthcare (5 mentions)
Superdex 75 column, by GE Healthcare (4 mentions)
Dawn heleos 2 mals detector, by Wyatt Technology (4 mentions)
Optilab t rex differential refractometer, by Wyatt Technology (4 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (4 mentions)
Superdex 75 10 300 column, by GE Healthcare (4 mentions)
Minidawn treos instrument, by Wyatt Technology (3 mentions)
Superdex 200 increase column, by GE Healthcare (3 mentions)
Viscotek tda 305 triple array detector, by Malvern Panalytical (3 mentions)
Omnisec, by Malvern Panalytical (3 mentions)
Hmw calibration kit, by GE Healthcare (3 mentions)
Prominence rf 20a, by Shimadzu (3 mentions)
Unicorn 5, by GE Healthcare (3 mentions)
Pd 10 column, by GE Healthcare (3 mentions)
Mono q 5 50 gl, by GE Healthcare (2 mentions)
Superdex 200 10 30 gl column, by GE Healthcare (2 mentions)
Carbonic anhydrase, by Merck Group (2 mentions)
169 protocols using kta purifier
1
Purification and Modification of MAbs
2
Purification of SARS-CoV-2 RBD Proteins
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Escherichia coli cells carrying the recombinant plasmids encoding for CΔ116-MrNV-CPβ-RBD or CΔ116-MrNV-CPδ-RBD were inoculated into 50 mL LB broth containing ampicillin (100 μg/mL) and cultured overnight at 37 °C. An aliquot (10 mL) of overnight culture was transferred to 1 L LB broth and incubated at 37 °C until the culture reached an OD600 of ~0.6. Protein expressions were induced by adding 1 mM IPTG into the culture, and it was incubated at 25 °C for 5 h. The cell pellets were harvested by centrifugation at 8000× g for 8 min. Purification of recombinant proteins was modified from a previous study by Kumar et al. [21 (link)], using a fast-protein liquid chromatography (FPLC) system (Äkta Purifier; GE Healthcare, Uppsala, Sweden). The HiTapTM SP HP 1 mL column (GE Healthcare, Buckinghamshire, UK) was washed with 10 column volume (CV) of binding buffer (50 mM HEPES, 100 mM NaCl, pH 7.4). The lysates were loaded onto the column, and bound proteins were eluted via a gradient of NaCl concentration (50 mM HEPES, 1 M NaCl, pH 7.4) at a flow rate of 1 mL/min and fractionated using a Frac-950 collector. The eluted proteins were then analyzed with SDS-PAGE and Western blotting.
3
Purification of Truncated PARN Proteins
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The plasmid containing the cDNA sequence of the wild type human PARN was kindly provided by Professor Anders Virtanen (Uppsala University, Sweden). The truncated proteins p60 (residues 1–520 AA) and p46 (residues 1–446 AA) were constructed by standard protocols of mutagenesis using the following primers: p60-forward, 5′-CGATGTCACATATGGAGATAATCAGGAGC-3'; p60-reverse 5′-GATCCTCGAGCTACTTCTCTTCCTGTTTTC-3'; p46-forward, 5′-GCTACTCGAGCTTCTCTTCCTGTTTTC-3'; and p46-reverse, 5′-GATCGTCGACTTAATGATCACGTTTAGGCTGC-3'. The obtained genes were cloned to the expression vector pET-28a (Novagen) and verified by sequencing. The recombinant proteins were overexpressed in Escherichia coli BL21 (DE3) (Stratagene, Heidelberg, Germany) and purified as described previously [18 (link),28 (link)]. In brief, the expression of the proteins was induced by 0.1 mM IPTG at 16 °C for 24 h. The proteins were isolated from the supernatant fraction of cell lysates by Ni2+-affinity chromatography (GE Healthcare),. The final products were purified using a Superdex 200 10/30 GL column equipped on an ÄKTA purifier (GE Healthcare). The protein concentration was determined according to the Bradford method [29 (link)]. The protein solutions used for analysis were prepared in buffer A containing 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM DTT, 0.2 mM EDTA and 20% (v/v) glycerol.
4
Size Exclusion Chromatography of Proteins
5
SDS-PAGE and Western Blot Analysis
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SDS-PAGE and Western transfer experiments were performed by standard procedures [30 (link)]. The immunoligands were detected applying mouse anti-penta-His (Qiagen) and secondary horse radish peroxidase-conjugated goat anti-mouse IgG antibodies (Dianova). Gel filtration chromatography was performed on an ÄKTA purifier (GE Healthcare) employing Superdex 200 10/300 GL column (GE Healthcare). Phosphate buffered saline (PBS) was used as running buffer at a constant flow rate of 0.7 ml/min. 100–500 μg protein were loaded in a volume of 0.5 ml PBS. Ferritin (440 kDa), human IgG1 (150 kDa), Conalbumin (75 kDa) and Ribonuclease A (13.7 kDa) were used for calibration. Data were analyzed with Unicorn 5.1 software (GE Healthcare).
6
Scalable Production of Purified AAV Vectors
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AAV vectors were produced by two‐plasmid (pDG1‐rs) cotransfection in HEK 293 cells essentially as described (Mietzsch et al, 2014 ). AAV vector backbones were packaged in serotype 1 capsids and HPLC‐purified from benzonase‐treated, cleared freeze–thaw supernatants by one‐step AVB sepharose affinity chromatography using 1 ml prepacked HiTrap columns on an ÄKTA purifier (GE Healthcare). Peak fractions were dialyzed against PBS and the titers of the highly purified AAV preparations were determined by qPCR, measured as AAV DNA‐containing genomic particles (gp) per ml, as described before (Mietzsch et al, 2014 ).
7
Peptide Trypsin Resistance Assay
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The resistance of the peptides to degradation by trypsin was assessed following the protocol of Bagheri and Hancock [15 ], with some modifications. Fresh trypsin solution was prepared by resuspending the lyophilized enzyme (11900 units/mg) in ammonium bicarbonate buffer ((NH4)HCO3 100 mM, pH 8.2) to 2.4 × 103 units/mL. Peptides (10 µM) in the same enzyme buffer were incubated with trypsin (30 units). Samples (0.4 mL) were taken at times 0, 15, 30, and 60 min for the peptide tritrpticin, while for all other peptides, only 0 and 60 min samples were collected. The trypsin digestion was stopped by addition of acetic acid/ trifluoroacetic acid (TFA) 2.5% solution (Sigma Aldrich, St. Louis, MO, USA) to 20% (v/v) at each time point. All samples were then stored at −20 °C until they were analyzed by reverse-phase chromatography using a 5C18-AR-300 Cosmosil column (Nacalai Tesque, Inc., Tokyo, Japan) with an acetonitrile/water gradient supplemented with TFA 0.05% (v/v) in an ÄKTA purifier (GE Healthcare, Pittsburg, PA, USA).
8
Purification of Protein Extract
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Concentrated supernatant from the culture of the strain X-33/pPic-clavMO containing 5 mg/mL of protein extract was lyophilized and injected into a size exclusion chromatography Äkta purifier (GE Healthcare). The column utilized was the Hiload™ 16/60 Superdex™ 75 prep grade column (GE Healthcare). This was equilibrated with filtered and degassed water MilliQ at room temperature. The flow rate utilized was 0.8 mL/min. Fractions were eluted with 5 mL volumes, totaling 22 fractions. These were monitored at 216 and 280 nm for 270 min. The fractions from 16 to 22 were lyophilized and used for further antibacterial assays.
9
Enzyme Purification Protocol using Affinity Chromatography
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For purification, the frozen cells were resuspended in sodium phosphate buffer (50 mM, pH 7.5, optionally supplemented with DNase) at RT and disrupted via sonication on ice. The resulting crude extract was centrifuged and the supernatant/cell-free extract (CFE) diluted to a protein concentration of ∼20 mg/mL. The diluted CFE was then precipitated at an ammonium sulfate (AS) concentration of 55% (w/v) and collected by centrifugation (18,600×g, 15 min, 4 °C). The resulting pellet was resuspended in sodium phosphate buffer (50 mM, 500 mM NaCl pH 7.5) and subjected to an affinity chromatography purification on a 5 mL HF HisTrap column on an Äkta purifier (GE Healthcare). Fractionated elution was carried out with imidazole (250 mM). The buffer was removed by a second AS precipitation with 65% AS. The resulting pellet was resuspended in ice-cold Tris/HCl. After the addition of sucrose (20 mg/mL) for stabilization during storage, the enzyme was shock frozen in liquid nitrogen and stored at −80 °C (van Beek et al. 2015 (link)).
The PTDH was purified using Ni Sepharose High Performance (GE Healthcare) as described before (Dudek et al. 2013 (link)). Ten percent glycerol (v/v) was added to the purified enzyme, which was shock frozen and stored at −20 °C.
The PTDH was purified using Ni Sepharose High Performance (GE Healthcare) as described before (Dudek et al. 2013 (link)). Ten percent glycerol (v/v) was added to the purified enzyme, which was shock frozen and stored at −20 °C.
10
Purification of Human Plasma Antithrombin
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Human plasma α-antithrombin was purified from a pool of 100 healthy subjects by heparin affinity chromatography on HiTrap Heparin columns (GE Healthcare, Barcelona, Spain), using an ÄKTA Purifier (GE Healthcare, Barcelona, Spain) in 50 mM Tris-HCl, pH 7.4 in a gradient from 0 to 3 M NaCl. Fractions with antithrombin were applied to a HiTrap Q column (GE Healthcare, Barcelona, Spain). Finally, proteins were eluted using a gradient from 0 to 1 M NaCl and desalted using dialysis tubing (Thermo Fisher, Spain). Recombinant antithrombin from media harvests was purified using the same strategy described for plasma antithrombin. Purity of proteins was evaluated by 8% SDS-PAGE by silver staining, as indicated elsewhere [17 (link)]. Proteins were stored at -70°C.
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