HEK293 cells were transfected with the appropriate expression vectors using Lipofectamine 3000 reagent (Invitrogen) and selected in DCM containing 500 μg/mL Geneticin (G418) to generate stable cell lines HEK293-hLiTCo, HEK293-hLiTCo-Albu, HEK293-mLiTCo and HEK293-mLiTCo-Albu. Conditioned media were collected and purified using Strep-Tactin purification system (IBA Lifesciences) using an ÄKTA Go system (Cytiva). The purified antibodies were dialyzed overnight at 4°C against PBS Phosphate-Buffered Saline (PBS, Corning) with 150 mM NaCl at pH 7.0, analyzed by 12% SDS-PAGE under reducing conditions and stored at 4°C. Size exclusion chromatography was performed on a Superdex 200 Increase 10/300 GL column (Cytiva) on and AKTA-GO chromatography system (Cytiva). 50 μL of the solutions at 0.15 (mLiTCo) or 0.07 g/L (mLiTCo-Albu) were injected into the column and run at room temperature in PBS pH 7.4, with a flow rate of 0.5 mL/min.
Kta go system
Manufactured by Cytiva
Sourced in United States
The ÄKTA go system is a versatile, compact chromatography system designed for fast and efficient protein purification. It features an intuitive user interface, automatic fraction collection, and advanced data analysis capabilities to support a wide range of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Imidazole, by Qiagen (2 mentions)
Ni nta agarose bead, by Qiagen (2 mentions)
Hitrap desalting column, by Cytiva (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Superdex 200 increase 10 300 gl column, by Cytiva (1 mentions)
Pbs phosphate buffered saline pbs, by Corning (1 mentions)
Strep tactin purification system, by IBA Lifesciences (1 mentions)
Pierce high capacity endotoxin removal spin column, by Thermo Fisher Scientific (1 mentions)
Histrap hp his tag protein purification columns, by Cytiva (1 mentions)
Pierce chromogenic endotoxin quant kit, by Thermo Fisher Scientific (1 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
Superdex 200 10 300 gl column, by Cytiva (1 mentions)
Cellfectin 2 reagent, by Thermo Fisher Scientific (1 mentions)
Imidazole, by Cytiva (1 mentions)
Type 50.2 ti rotor, by Beckman Coulter (1 mentions)
5 protocols using kta go system
1
Generation and Purification of Recombinant Antibodies
2
MSAD-1 Protein Purification from M. abscessus
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Recombinant MSAD-1 protein of M. abscessus (NCBI accession number OLT57519.1 ) were purified from Escherichia coli as previously described with minor modification (Jeong et al., 2022 (link)). Briefly, the DNA sequence of MSAD-1 was amplified from M. abscessus ATCC 19977T using PCR with following primer sets (forward primer, 5′-TTT GGA TCC ATG CCA TTG GTG CGC ATC GAC CTC-3′; reverse primer, 5′-AAA AAG CTT GTG CGC CTG CGG CGG GCA C-3′), and cloned into pET-28a. The expression and purification of MSAD-1 were commercially commissioned by Bionics (Seoul, Republic of Korea). In detail, the protein expression was induced in E. coli Rosetta2 (DE3) strains (Novagen, WI, USA) transformed with pET28a-MSAD-1 by adding 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 26°C for 6 h. Cultured bacterial cells were harvested and sonicated for 30 cycles at 70% amplitude. After centrifuge, the supernatant was purified with HisTrap™ HP His tag protein purification columns (Cytiva, MA, USA) for Ni-NTA affinity chromatography via ÄKTA go system (Cytiva, MA, USA). Purified proteins were subjected to endotoxin removal using Pierce™ high-capacity endotoxin removal spin columns (Thermo Scientific, MA, USA) and quantified by Pierce™ chromogenic endotoxin quant kit (Thermo Scientific, MA, USA).
3
Gel Filtration Chromatography of Proteins
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Gel filtration chromatography was performed using the ÄKTA go™ system and Superdex 200 10/300 GL column (Cytiva, MA, USA). Injection of protein solution into a column and subsequent elution were performed with isocratic conditions. All experiments were performed at 25 °C at a flow rate of 0.75 ml min−1 in a buffer containing 150 mM NaCl, 0.1 mM DTT, 1 mM EDTA, and 25 mM Tris–HCl (pH 7.5). HPLC–UV chromatograms were recorded at 280 nm. After centrifugation, 300 μL of sample solutions was injected, and sufficient ultraviolet (UV) absorption was obtained. Gel filtration standard (Bio-Rad Laboratories, Inc.) was used as molecular weight standards.
4
Recombinant Expression and Purification of CAMSAP2
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Human CAMSAP2 was recombinantly expressed as a fusion protein with N-terminally tagged 6His-GFP in a pFastBac vector (pFastBac+GFP-CAMSAP2 was a gift from Ron Vale (Addgene plasmid # 59037; http://n2t.net/addgene:59037 ; RRID:Addgene_59037) (Hendershott and Vale, 2014 (link))). Baculovirus was produced in Sf21 insect cells after transfection with cellfectin II reagent (Thermo Fisher Scientific). Infected Sf21 cells were incubated at 27 °C for 60 h, followed by harvesting via centrifugation (800 × g for 3 min), flash freezing and storage at –80 °C until protein purification. The insect cell pellet was resuspended in lysis buffer (50 mM HEPES pH 7.6, 300 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.1% Triton X-100, cOmplete EDTA-free protease inhibitor cocktail (Roche) and PMSF), sonicated for 3 × 1 min with 0.6 amplitude and 0.5 cycles (Hielscher UP50H) and centrifuged for 30 min at 20,000 × g and 4 °C. The supernatant was incubated with Ni-NTA Agarose beads (Qiagen) for 2 h at 4 °C, washed with lysis buffer and eluted with lysis buffer supplemented with 300 mM imidazole (Roth). imidazole was removed via buffer exchange through a HiTrap Desalting column (5 mL, Cytiva) on an ÄKTA go system (Cytiva). Proteins were flash-frozen and stored at −80 °C until usage.
5
Recombinant Ran(Q69L) Protein Purification
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Ran(Q69L), cloned into a pQE32 vector, was transformed into Escherichia coli competent cells (strain BL21-CodonPlus(DE3)-RIL). Expression was induced with 0.2 mM IPTG (Roth) at an optical density of 0.5–0.8 in 2xYT medium. Cells were grown at 30 °C for 16 h. Afterwards, cells were collected, washed with PBS and cell pellet was stored at −80 °C. The pellet was resuspended in lysis buffer (10 mM HEPES pH 7.6, 100 mM KCl, 1 mM MgCl2, 5% v/v glycerol, 1 mM DTT, 0.1% Triton X-100, cOmplete EDTA-free protease inhibitor cocktail (Roche) and PMSF), lysed by sonification and clarified by centrifugation at 40,000 rpm for 20 min in a Type 50.2 Ti Rotor (Beckman Coulter). Supernatant was incubated with Ni-NTA Agarose beads (Qiagen) for 2 h at 4 °C, washed with lysis buffer and eluted with lysis buffer supplemented with 300 mM imidazole (Roth). imidazole was removed via buffer exchange through a HiTrap Desalting column (5 mL, Cytiva) on an ÄKTA go system (Cytiva) operated using the UNICORN software (version 7.6). Ran(Q69L) was concentrated to 350 μM and stored at −80 °C.
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