cDNA encoding a truncated human ACE2 fused to human albumin was sub-cloned into pFUSE2ss-CLIg-hk (InvivoGen). The vector was transiently transfected into Expi293F cells in suspension (Thermo Fisher Scientific) using the ExpiFectamine 293 transfection kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were cultured for 7 days at 37 °C with 80% humidity and 8% CO2 on an orbital shaker platform set to 125 rpm before the medium was collected. The secreted fusion protein was purified on a CaptureSelect™ human albumin affinity matrix (Life Technologies), and protein eluted by adding 20 mM Tris and 2.0 M MgCl2, pH 7.0 before up-concentration using Amicon® Ultra-15 50K Centrifugal Filter Units (Merck Millipore). Buffer exchange to PBS was performed before size exclusion chromatography (Äkta Avant, GE Healthcare) with a SuperdexTM 200 Increase 10/300 GL (Cytiva) prior to up-concentration using Amicon® Ultra-0.5 Centrifugal Filter Units (Merck Millipore). The protein eluted as a dimer. For hapten-conjugation, digoxigenin-NHS (Sigma Aldrich, cat. No 11333054001, 40μg/mg protein) was added to protein solublized in PBS. After 30 min of incubation at 22 °C, free digoxigenin was removed using Amicon® Ultra-0.5 Centrifugal Filter Units.
Kta avant
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden
The ÄKTA avant is a versatile and automated chromatography system designed for a wide range of purification applications. It features flexible software, intuitive user interface, and advanced monitoring capabilities to support efficient and reliable purification processes.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions)
Pfuse2ss clig hk, by InvivoGen (1 mentions)
Amicon ultra 0.5 centrifugal filter unit, by Merck Group (1 mentions)
Superdextm 200 increase 10 300 gl, by Cytiva (1 mentions)
Digoxigenin nhs, by Merck Group (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
Hitrap desalting column, by GE Healthcare (1 mentions)
Histrap excel column, by GE Healthcare (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Ceramic hydroxyapatite type 1 resin, by Bio-Rad (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Hitrap desalting 5 ml column, by GE Healthcare (1 mentions)
Quantichrom bcg albumin assay kit, by BioAssay Systems (1 mentions)
Uvmc2 spectrophotometer, by Safas (1 mentions)
11 protocols using kta avant
1
Production of Digoxigenin-Labeled Truncated ACE2
2
Size Exclusion Chromatography for Trivalent Protein Oligomers
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A Superdex 200 Increase 10/300 GL column attached to the ÄKTA avant (GE Healthcare) system was used for size exclusion chromatography (SEC) to detect and separate the oligomeric populations of the purified trivalent proteins. The column was equilibrated with 1 × PBS, pH 7.4. Then, 500 µL of concentrated trivalent protein was loaded onto the column. Proteins were eluted at a flow rate of 0.5 mL/min, and the protein signals were monitored by measuring the ultraviolet light absorbance at 280 nm. The contents of different oligomeric fractions were evaluated by the peak integration area. The trimeric fractions were collected for further characterization.
3
Production and Purification of Angiogenic Proteins
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The recombinant VEGF-A109, VEGF-A165, VamminKPRR and VamminPRRK proteins were produced in adherent HEK293T cells by calcium phosphate transfection with plasmids encoding N-terminally His-tagged proteins. On the following day, DMEM (Sigma-Aldrich) supplemented with 10% FBS was replaced with serum free media and cells were incubated for 2 days. The proteins were purified using HisTrapExcel columns with ÄktaAvant (GE Healthcare) using gradient elution from 0 to 500 mM Imidazole in PBS. The collected fractions were analyzed by SDS-PAGE. Pure fractions were pooled, diluted to PBS and concentrated using Amicon Ultra-15 3 K Centrifugal Filter Units and final buffer exchange to PBS was performed using HiTrap Desalting columns (GE Healthcare). Protein concentration was assessed using a BCA Protein Assay Kit (Thermo Scientific) and verified by SDS-PAGE. Recombinant Vammin, sVEGFR2-Fc and sNRP1-Fc were produced as described earlier17 (link).
4
Purification of Recombinant Human SGSH
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rhSGSH was purified from cell culture fluid using either a two or three step purification using an ÄKTA avant (GE Healthcare). Filtered harvest was diluted at a 1:2 volume ratio with 20 mM Tris, pH 7.3 and loaded onto a Capto Q ion exchange resin (GE Healthcare) and eluted with 250 mM NaCl. The Capto Q eluate was then adjusted to 20 mM Tris, 1 M ammonium sulfate, pH 7.3, and loaded onto a Capto Butyl hydrophobic interaction chromatography resin (GE Healthcare). Elution was generated by a linear gradient of declining ammonium sulfate. For purifications with three steps, the Capto Butyl eluate was diluted 6-fold with 2 mM phosphate, 20 mM Tris, pH 6.2, and loaded onto a ceramic hydroxyapatite type I resin (Bio-Rad Laboratories). Elution was achieved with a 50 mM phosphate, 20 mM Tris, 150 mM NaCl, pH 7.5 buffer. rhSGSH was formulated into artificial CSF (3 mM KCl, 5 mM phosphate, 150 mM NaCl, pH 7.3) and concentrated to 29.6 mg/ml for the two-step Lot 1 and 32.2 mg/ml for the three-step Lot 2 by ultrafiltration/diafiltration using a 30 kDa molecular weight cutoff polyethersulfone membrane. All lots were greater than 95% pure (RP-HPLC, Agilent C3 Zorbax 300SB) and within an endotoxin specification of ≤0.05 EU/mg rhSGSH (Endosafe PTS) whether the two-step or three-step purification was used.
5
Isolation of S100A8/A9 protein complexes
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For the isolation of S100A8, S100A9, and hetero/homo-complexes from the cytosolic fraction, we adapted a previously described method [25 (link)]. In short, 50% (w/v) ammonium sulphate (AS) was added to the cytosolic fraction and incubated on ice for 1 hour. After centrifugation at 10,000× g for 30 min at 4 °C, supernatant was dialyzed using a 3.5 K MWCO Slide-A-Lyzer Dialysis Cassette (Thermo Fisher Scientific, Waltham, MA, USA) against “buffer A” (1 mM EDTA and 50 mM Tris-HCl in demi water, pH 8.5) at 4 °C. During the first and second hours of dialysis, buffer A contained 25% and 12.5% (w/v) AS, respectively, before dialyzing against 0% (w/v) AS overnight.
Anion exchange chromatography (AEX) was performed using the ÄKTA avant (GE Healthcare, Chicago, IL, USA) and a 1-mL HiTrap MonoQ AEX column (GE Healthcare). A salt gradient of 0 to 1 M with buffer A and “buffer B” (buffer A + 1 M NaCl) in 20-column volumes (CVs) was used to elute proteins. Fractions containing at least 100 µg/mL of S100A8/A9 (as determined by ELISA detecting the S100A8/A9 hetero-complex) were pooled and dialyzed against PBS using a 3.5 K MWCO Slide-A-Lyzer Dialysis Cassette to remove buffer A.
Anion exchange chromatography (AEX) was performed using the ÄKTA avant (GE Healthcare, Chicago, IL, USA) and a 1-mL HiTrap MonoQ AEX column (GE Healthcare). A salt gradient of 0 to 1 M with buffer A and “buffer B” (buffer A + 1 M NaCl) in 20-column volumes (CVs) was used to elute proteins. Fractions containing at least 100 µg/mL of S100A8/A9 (as determined by ELISA detecting the S100A8/A9 hetero-complex) were pooled and dialyzed against PBS using a 3.5 K MWCO Slide-A-Lyzer Dialysis Cassette to remove buffer A.
6
Analytical SEC Protein Purification
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Analytical SEC was conducted at room temperature on a Superdex200 10/300 GL column (GE Healthcare Life Sciences) connected to the ÄKTAavant (GE Healthcare Life Sciences) system operated at 0.5 ml/min. The UV absorbance at 260 and 280 nm was monitored. Protein purified according to protocol 1 or protocol 2 (1 mg/ml) was injected in a volume of 0.2 ml.
7
Purification of L-asparaginase using HiTrap
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HiTrap® Desalting 5 ml column (GE Healthcare, United Kingdom) connected with ÄKTA avant (GE Healthcare, United Kingdom) was used for the removal of 10% (v/v) glycerol and to exchange the buffer of refolded and purified L -asparaginase with 20 mM Tris–HCl. For that, the column was washed with five column volumes of water. The column was equilibrated with two column volumes of 20 mM Tris–HCl; 1.5 ml of refolded L -asparaginase in 50 mM Tris–HCl and 10% (v/v) glycerol at pH 8.5 was loaded onto the column and subsequently eluted. The eluted fractions were collected and pooled. Washing, equilibration, and elution were performed at a flow rate of 4 ml/min.
8
Optimizing Antibody Purification and Concentration
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Example 2
Concentrated (100 g/L) monoclonal antibodies (mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6) were each purified on an Äkta avant (GE Healthcare Life Sciences) through a XK26/100 Superdex 200 pg column in 10 mM MES pH 6.0 50 mM sodium chloride. The monomer mAb fraction was collected and concentrated using an Easy-Load MAsterFlex L/S (Cole Parmer) pump in tandem with a VivaFlow 200 30,000 MWCO HY membrane. 150 mL of each concentrated antibody was split in to 3 fractions and loaded in to a 10,000 MWCO dialysis cassette and exchanged against 2 L of 10 mM MES pH 6.0, 250 mM sodium chloride; 10 mM MES pH 6.0, 50 mM sodium chloride; and 10 mM MES pH 6.0, 10 mM sodium chloride solutions. The solutions were concentrated, using a centrifugal filter unit with a 50,000 MWCO, to a final volume of approximately 15 mL in their respective buffers. The concentrations were measured using a SoloVPE (C Technologies, Inc.). Antibody could not be concentrated beyond nominally 60 g/L in 10 mM MES pH 6.0, 10 mM sodium chloride. The two other salt concentrations (250 mM and 50 mM sodium chloride) were adjusted to nominally 80 g/L. An aliquot from the three conditions were taken and diluted to 10 g/L. A sample of 80 g/L, 10 g/L and buffer were affixed to a CG-MALS device (Wyatt Technology) and the light scattering signal was measured as a function of mAb concentration.
9
Gel Filtration of Plasma/Serum Proteins
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Citrate-plasma or serum (500 μL) pooled from 10 healthy individuals was applied to a Superose 6 10/300 GL column connected to a ÄKTA AVANT (GE Healthcare, Uppsala, Sweden). Gel filtration chromatography was performed in TBS at a flow rate of 0.4 mL/min, and 30 fractions each containing 300 μL were collected. ApoM, S1P and albumin were measured in the fractions using methods described above and with the QuantiChrom BCG Albumin Assay Kit (Bioassay Systems, USA).
10
Purification of HaloTag Protein
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