Human codon-optimized Cas9 (Addgene plasmid 42230) was subcloned into a custom pET-based expression vector with an N-terminal hexahistidine (6×His) tag followed by a SUMO protease cleavage site. The fusion construct was transformed into E. coli Rosetta 2(DE3) competent cells (Millipore), grown in LB media to OD600 0.6, and induced with 0.2 mM IPTG for 16h at room temperature. Cells were pelleted, resuspended and washed with Milli-Q H2O supplemented with 0.2 mM PMSF, and lysed with lysis buffer (20 mM Trizma base, 500 mM NaCl, 0.1% NP-40, 2 mM DTT, 10 mM imidazole). The lysis buffer was supplemented with protease inhibitor cocktail (Roche) immediately prior to use. Whole lysate was sonicated at 40% amplitude (Biologics Inc., 2s on, 4s off) prior to ultracentrifugation (30,000 rpm for 45m). The clarified lysate was applied to cOmplete His-tag purification columns (Roche), washed with wash buffer 1 (20 mM Trizma base, 500 mM NaCl, 0.1% NP- 40, 2 mM DTT, 10% glycerol, 10 mM imidazole) and wash buffer 2 (20 mM Trizma base, 250 mM NaCl, 0.1% NP-40, 2 mM DTT, 10% glycerol, 50 mM imidazole). The 6xHis affinity tag was released via SUMO protease cleavage and bound protein was eluted with a linear gradient of 150 mM – 500 mM imidazole. Eluted protein was concentrated with Amicon centrifugal filter units with Ultracel membrane (Millipore) and stored at -80°C.
Complete his tag purification column
Manufactured by Roche
Sourced in United States, United Kingdom, Switzerland, Germany
The Complete His-Tag Purification Column is a pre-packed chromatography column designed for the purification of His-tagged proteins. It utilizes a high-capacity Ni-NTA resin to efficiently capture and purify the target protein from a complex sample.
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Lab products found in correlation
Bca method, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti c myc antibody beads, by Fujifilm (1 mentions)
Amicon ultra 10 000 nmwl, by Merck Group (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Kta avant 25 system, by GE Healthcare (1 mentions)
Hiscale 16 20 column, by GE Healthcare (1 mentions)
Ni sepharose high performance, by GE Healthcare (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Profinia protein purification system, by Bio-Rad (1 mentions)
Valproic acid, by Cayman Chemical (1 mentions)
Hiload 16 600 superdex 200 pg column, by GE Healthcare (1 mentions)
Histrap hp 5 ml column, by GE Healthcare (1 mentions)
Branched pei, by Merck Group (1 mentions)
Hiprep 26 10 desalting column, by GE Healthcare (1 mentions)
Enrich sec 650 column, by Bio-Rad (1 mentions)
Geneart gene synthesis service, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 medium, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Imidazol, by Merck Group (1 mentions)
15 protocols using complete his tag purification column
1
Recombinant Cas9 Protein Purification
2
Recombinant Protein Purification Protocols
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Myc-tagged CDS1, CDS2, PCYT1A, and PCYT1B, as well as Flag-tagged PCYT2 and TagD recombinant proteins, were produced by transfecting expression vectors in HCT116 cells. After 48–72 h of transfection, recombinant proteins were purified by anti-c-Myc Antibody Beads (10D11; Wako) or Anti-FLAG M2 affinity gel (Sigma-Aldrich) following the standard protocol. His6-tagged PCYT2α or PCYT2β recombinant protein was produced in E. coli BL21-CodonPlus (DE3) (Agilent Technologies, Santa Clara, CA, USA), purified by cOmplete His-Tag purification columns (Roche, Basel, Switzerland), and concentrated to 1 mg/mL using Amicon Ultra 10,000 NMWL (Merck). Recombinant α-DG 373(T322R)-Fc proteins were prepared from the culture supernatants of the transfected cells as described previously [20 (link)].
3
Purification of His-tagged and C-tagged Proteins
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From the harvested culture supernatants, HA proteins were purified by a two-step protocol using an ÄKTA Avant 25 system (GE Healthcare Life Sciences). For his-tagged proteins, the material was applied to either a prepacked cOmplete His-tag purification column (Roche) or self-packed HiScale 26/60 column with Ni Sepharose High Performance (GE Healthcare Life Sciences). Following a wash with 1 mM imidazole, the bound proteins were eluted with a step gradient to 300 mM imidazole. For C-tagged protein, the clarified supernatant was loaded on a HiScale 16/20 column (GE Healthcare) packed with an affinity resin that consisted of a C-tag–specific single domain antibody immobilized on Agarose-based beads (Thermo Fisher Scientific). The elution of the C-tagged proteins was performed using a Tris buffer containing 2 M MgCl2. The His-tag– and C-tag–containing elution fractions were pooled and filtered through a Millex-GV 0.22-µM filter membrane (Millipore Sigma). To further polish the purified protein, SEC was performed by running a HiLoad Superdex 200-pg 26/60 column (GE Healthcare Life Sciences). Peak fractions were analyzed on a sodium dodecyl-sulfate polyacrylamide gel electrophoresis, pooled, and, when the concentration was <1 mg/mL, concentrated by centrifugation using Amicon Ultra-15 centrifugal filters with a 10-kDa cutoff.
4
Dicer-SIRT7 Interaction Assay
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Purified recombinant human Dicer was incubated with His-tagged recombinant SIRT7 in binding buffer (50 mM NaH2PO4, pH8.0, 300 mM NaCl) for 3 h. BSA was used to compensate the missing protein when only one protein (Dicer or SIRT7) was included in the assay. The mixture was applied to a Complete His-Tag Purification Column (Roche, Mannheim, Germany) and incubated for 10 min. The column was then washed with 10 column volumes of binding buffer to remove the unbound proteins, and the bound proteins were eluted with a buffer containing 50 mM NaH2PO4 (pH8.0), 300 mM NaCl and 250 mM imidazole. Representative unbound and bound fractions were subjected to western blot.
5
Purification of Notch Protein Constructs
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Notch ligand and receptor constructs were recombinantly expressed in S2 insect cells (Expres2ion® Biotechnologies, Denmark) as C‐terminally His‐tagged fusion proteins. Media containing recombinantly expressed protein were filtered and loaded onto a cOmplete His‐tag Purification Column (Roche Diagnostics, UK), for purification via His‐tag. Following washing with 50 mM Tris pH 9.0, 5 mM imidazole pH 8.0, 200 mM NaCl and 1 mM CaCl2, proteins were eluted with buffer containing 250 mM imidazole pH 8.0. Following overnight dialysis, proteins were further purified by size‐exclusion chromatography (SEC) using a Superdex S200 (ligands) or S75 (receptors) preparative column in 20 mM Tris pH 7.5, 200 mM NaCl and 1 mM CaCl2.
6
Purification of TR4-LBD Protein
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The TR4-LBD expression
plasmid (pSumo-TR4-LBD) was kindly provided by Dr. Zhou from the School
of Medicine, Michigan University. TR4-LBD was heterologously expressed
in BL21 (DE3). The resulting proteins tagged with a His6-sumo-motif
at their N-termini were isolated and purified using a cOmplete His-tag
purification column (Roche, Mannheim, Germany). The His-sumo-motif
was then removed using the RobustCutter Sumo protease (robustnique,
Tianjin, China). Another cOmplete His-tag purification was performed
to collect the TR4-LBD protein in the flowthrough. The protein was
changed to suitable buffers for the following SPRi (20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic
acid (HEPES), 150 mM NaCl, pH 7.5) and Circular Dichroism (5 mM phosphate
buffer) assay using the PD-10 desalting column (GE Healthcare, Freiburg,
Germany). Protein concentrations were determined using the BCA method
(ThermoFisher Scientific, Rockford).
plasmid (pSumo-TR4-LBD) was kindly provided by Dr. Zhou from the School
of Medicine, Michigan University. TR4-LBD was heterologously expressed
in BL21 (DE3). The resulting proteins tagged with a His6-sumo-motif
at their N-termini were isolated and purified using a cOmplete His-tag
purification column (Roche, Mannheim, Germany). The His-sumo-motif
was then removed using the RobustCutter Sumo protease (robustnique,
Tianjin, China). Another cOmplete His-tag purification was performed
to collect the TR4-LBD protein in the flowthrough. The protein was
changed to suitable buffers for the following SPRi (20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic
acid (HEPES), 150 mM NaCl, pH 7.5) and Circular Dichroism (5 mM phosphate
buffer) assay using the PD-10 desalting column (GE Healthcare, Freiburg,
Germany). Protein concentrations were determined using the BCA method
(ThermoFisher Scientific, Rockford).
7
Recombinant Protein Expression and Purification
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Escherichia coli strain KRX (Promega, Madison, WI, USA) was transformed with GmIOMT1 (pET-53-DEST) and precultured in 20 ml of liquid LB medium (50 mg l−1 carbenicillin) at 37�C overnight. Two milliliters of pre-culture and final concentration of 0.15% glucose, 0.1% rhamnose and 0.5 mM IPTG were added into a 500-ml Erlenmeyer flask containing 200 ml of the same medium and cultured at 16�C overnight. The cell pellet was collected by centrifuge, resuspended in lysis buffer (300 mM KCl, 50 mM KH2PO4, 5 mM imidazole, pH 8.0) and then disrupted by sonication. The supernatant collected by centrifuge and His-tagged protein was purified using Profinia protein purification system (Bio-Rad, M�nchen, Germany) with Profinia IMAC Purification Kit (Bio-Rad) and cOmplete His-Tag Purification Column (Roche Diagnostics, Rotkreuz, Switzerland).
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Escherichia coli strain KRX (Promega, Madison, WI, USA) was transformed with GmIOMT1 (pET-53-DEST) and precultured in 20 ml of liquid LB medium (50 mg l−1 carbenicillin) at 37�C overnight. Two milliliters of pre-culture and final concentration of 0.15% glucose, 0.1% rhamnose and 0.5 mM IPTG were added into a 500-ml Erlenmeyer flask containing 200 ml of the same medium and cultured at 16�C overnight. The cell pellet was collected by centrifuge, resuspended in lysis buffer (300 mM KCl, 50 mM KH2PO4, 5 mM imidazole, pH 8.0) and then disrupted by sonication. The supernatant collected by centrifuge and His-tagged protein was purified using Profinia protein purification system (Bio-Rad, M�nchen, Germany) with Profinia IMAC Purification Kit (Bio-Rad) and cOmplete His-Tag Purification Column (Roche Diagnostics, Rotkreuz, Switzerland).
8
Purification of Recombinant Human IL-18BP
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Adherent HEK293S MGAT1-/- cells (68 (link)) were grown in 5-layer cell culture flasks (Falcon Multi-Flask, Corning) in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Life Technologies, Thermo Fisher Scientific), supplemented with 10% fetal calf serum (Bodinco). Upon transfection, the growth medium was exchanged for DMEM supplemented with 3.6 mM valproic acid (item 13033, Cayman Chemical Company). Transient expression of human IL-18BP or IL-18BPΔN was achieved using branched PEI (Mn ∼ 10000, Cat.:40872-7, Sigma-Aldrich) as transfection reagent (67 (link)). After 4 days of expression, filtered conditioned medium was loaded onto a 5 ml cOmplete His-tag Purification Column (Roche Diagnostics). The protein was eluted with 250 mM imidazole in HBS after which the imidazole was removed using a HiPrep 26/10 Desalting column (GE Healthcare). The Avi-His6-tag was cleaved by caspase-3 (produced in-house) and the glycans were trimmed by Endo H (produced in-house) at 37 °C for 1 h. The flow-through of the HisTrap HP 5 ml column (GE Healthcare) containing the cleaved protein was concentrated and injected onto a HiLoad 16/600 Superdex 200 pg column (GE Healthcare Life Sciences) using HBS as running buffer. Fractions containing human IL-18BP or IL-18BPΔN were pooled, flash frozen, and stored at −80 °C. The purity of the protein was evaluated on SDS-PAGE stained with Coomassie.
9
Purification of Recombinant PahZ1KP-2 Enzyme
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PahZ1KP-2 was expressed using the recombinant E. coli BL21(DE3) harboring pPAA1KP-2, in which the enzyme was designed as a His-tagged fusion protein, as was reported in our previous work (Hiraishi et al. 2009 (link)). PahZ1KP-2 was purified from the soluble fraction of the recombinant E. coli with a cOmplete His-Tag Purification Column (Roche Applied Science). After the sample solution was applied, the column was washed with buffer A (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The enzyme was eluted with buffer B (50 mM NaH2PO4, 300 mM NaCl, and 150 mM imidazole, pH 7.4). The enzyme fractions were collected, concentrated, and exchanged with 10 mM phosphate buffer (pH 7.0). The purity and concentration of the purified PahZ1KP-2 were determined by SDS-PAGE and the Bradford method, respectively (Laemmli 1970 (link); Bradford 1976 (link)).
10
Production and Purification of HIV-1 Env Trimers
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HIV-1 Env and sCD4 D1D2 His-Tagged genes (codon optimized for Homo sapiens expression) were either created using published sequences or designed in silico, and cloned into pcDNA3.1(+) using GeneArt gene synthesis service (ThermoFisher Scientific). pConS gp160 clone was donated by David Montefiori. sCD4 D1D2 His-Tag was produced by transfecting 293T.17 cells using polyethyleneimine (PEI) (Polysciences) and purified on a cOmplete® His-Tag purification column (Roche), following the manufacturer’s instructions, and stored in PBS at −20°C.
Env soluble trimers ConSOSL.UFO.664 and ConSOSL.UFO.664 Myc-HIS tagged version were produced in 293T.17 using triple layered T-175 flasks. After transfection, FreeStyle 293 medium (GIBCO) was added (90 mL per flask) and harvested 48h later. Cellular debris were pelleted and the supernatant filtered (0.45 μm). The trimers were concentrated using 100kDa MWCO Amicon ultrafiltration columns (Merck Millipore) and transferred in PBS. Trimers were then purified by size exclusion chromatography (SEC) on an NGC medium pressure liquid chromatography (MPLC) system (BioRad) using an Enrich SEC 650 column (BioRad). The collected trimer fractions were then concentrated using ultrafiltration columns, aliquoted and stored at −80°C for further analysis.
Env soluble trimers ConSOSL.UFO.664 and ConSOSL.UFO.664 Myc-HIS tagged version were produced in 293T.17 using triple layered T-175 flasks. After transfection, FreeStyle 293 medium (GIBCO) was added (90 mL per flask) and harvested 48h later. Cellular debris were pelleted and the supernatant filtered (0.45 μm). The trimers were concentrated using 100kDa MWCO Amicon ultrafiltration columns (Merck Millipore) and transferred in PBS. Trimers were then purified by size exclusion chromatography (SEC) on an NGC medium pressure liquid chromatography (MPLC) system (BioRad) using an Enrich SEC 650 column (BioRad). The collected trimer fractions were then concentrated using ultrafiltration columns, aliquoted and stored at −80°C for further analysis.
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