The resulting plasmid was transformed into the Escherichia coli strain BL21 (DE3; Novagen, USA). The E. coli strain was cultured in 4 L of Terrific broth at 37°C until an OD600 of approximately 1.5, and protein expression was induced using 0.5 mM IPTG at 30°C. After cell harvest by centrifugation, cells were resuspended in a lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM 2-mercaptoethanol. The cells were disrupted using a continuous-type French press (Constant Systems, UK) at 23 kpsi pressure and was subjected to centrifugation at 19,000 × g for 30 min at 4°C to remove the cell debris. The supernatant was loaded onto an affinity chromatography column using a cobalt-charged agarose resin (Qiagen, The Netherlands) in lysis buffer. The target protein was eluted with lysis buffer supplemented with 250 mM imidazole and 0.5 mM EDTA. The eluate was treated with TEV protease and then loaded onto a HiTrap Q column (GE Healthcare, USA) to cleave the hexa-His-tag. A linear gradient of increasing NaCl concentration was applied to the HiTrap Q column. The fractions that contained the protein were applied onto a size exclusion chromatography column (HiLoad Superdex 200 26/600 column; GE Healthcare) pre-equilibrated with lysis buffer.
Hiload 26 600 superdex 200 column
Manufactured by GE Healthcare
Sourced in United States
The HiLoad 26/600 Superdex 200 column is a size exclusion chromatography column designed for the purification of proteins and other biomolecules. It features a packed bed of Superdex 200 resin, which enables the separation of molecules based on their size and shape. The column has a bed volume of 320 ml and a column diameter of 26 mm, making it suitable for a range of purification applications.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap q column, by GE Healthcare (2 mentions)
Continuous type french press, by Constant Systems (2 mentions)
Hitrap heparin column, by GE Healthcare (2 mentions)
Complete mini protease inhibitor, by Roche (2 mentions)
Glutathione conjugated agarose, by GE Healthcare (2 mentions)
Prescission protease, by GE Healthcare (2 mentions)
Hitrap hp column, by GE Healthcare (2 mentions)
Q sepharose, by GE Healthcare (1 mentions)
Q sepharose, by Bio-Rad (1 mentions)
Histrap hp, by GE Healthcare (1 mentions)
Resource q, by GE Healthcare (1 mentions)
Superdex 75 10 300 gl, by GE Healthcare (1 mentions)
Superdex 200 increase 3.2 300 column, by GE Healthcare (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Ds 11 spectrophotometer, by DeNovix (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Benzamidine, by Carl Roth (1 mentions)
Benzonase, by Merck Group (1 mentions)
Cyanocobalamin, by Merck Group (1 mentions)
Cobinamide, by Merck Group (1 mentions)
13 protocols using hiload 26 600 superdex 200 column
1
Purification of Recombinant Protein from E. coli
2
Purification and Characterization of Major Royal Jelly Protein 1
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Fresh royal jelly (RJ) samples were collected from Apis mellifera and Apis cerana colonies located in several regions of Zhejiang province during the flowering periods of different nectar plants. All RJ samples were stored at −20 °C until analyses.
One gram of RJ was dissolved in 10 mL of 40 mM phosphate buffer (pH 8.0). The suspension was centrifuged (25,000 g) for 15 min at 4 °C to separate the supernatant and pellet, and the supernatant was filtered with the 0.45 μm membrane filter. After adding 30 mL 20 mM Tris-HCl (pH 8.0), the filtered fluid was concentrated to 5 mL by ultrafiltration through a 30 kDa MW cutoff membrane (Millipore). The concentrated solution was further purified by the Q sepharose (Bio-rad). The protein was eluted with a linear gradient of NaCl from 0 to 1.0 M at a flow rate of 0.5 mL/min, and the elution was detected by SDS-PAGE and Coomassie blue staining. The fractions containing MRJP1 were collected and concentrated to 2 mL. MRJP1 collected from Q sepharose was applied to a Hiload Superdex-200 26/600 column (GE Healthcare) previously equilibrated with the buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The column was then eluted at a flow rate of 1.5 mL/min, and the fractions containing MRJP1 monomer and oligomer protein were collected, respectively.
One gram of RJ was dissolved in 10 mL of 40 mM phosphate buffer (pH 8.0). The suspension was centrifuged (25,000 g) for 15 min at 4 °C to separate the supernatant and pellet, and the supernatant was filtered with the 0.45 μm membrane filter. After adding 30 mL 20 mM Tris-HCl (pH 8.0), the filtered fluid was concentrated to 5 mL by ultrafiltration through a 30 kDa MW cutoff membrane (Millipore). The concentrated solution was further purified by the Q sepharose (Bio-rad). The protein was eluted with a linear gradient of NaCl from 0 to 1.0 M at a flow rate of 0.5 mL/min, and the elution was detected by SDS-PAGE and Coomassie blue staining. The fractions containing MRJP1 were collected and concentrated to 2 mL. MRJP1 collected from Q sepharose was applied to a Hiload Superdex-200 26/600 column (GE Healthcare) previously equilibrated with the buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The column was then eluted at a flow rate of 1.5 mL/min, and the fractions containing MRJP1 monomer and oligomer protein were collected, respectively.
3
Purification of Selenomethionine-Labelled Protein
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Transformed E. coli cells were cultured in 4 L of Terrific broth or M9 medium supplemented with L-(+)-selenomethionine at 37 °C. Protein expression was induced by 0.5 mM IPTG at 30 °C. After the cells were harvested by centrifugation, they were resuspended in lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM 2-mercaptoethanol. The cells were disrupted using a continuous-type French press (Constant Systems Limited, Daventry, UK) at 23 kpsi pressure, and the cell debris was removed by centrifugation at 19,000 × g for 30 min at 4 °C, after which the supernatant was loaded onto cobalt-Talon affinity agarose resin (Qiagen, The Netherlands) in lysis buffer. The target protein was eluted with lysis buffer supplemented with 250 mM imidazole. The eluate was treated with TEV protease to cleave the hexaHis-tag and was then loaded onto a HiTrap Q column (GE Healthcare, USA). A linear gradient of increasing NaCl concentration was applied to the HiTrap Q column. The fractions that contained the protein were applied onto a size exclusion chromatography column (HiLoad Superdex 200 26/600 column; GE Healthcare, California, USA) pre-equilibrated with lysis buffer.
4
Purification of Recombinant TCR Proteins
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Periplasmic fractions from large-scale expression were purified by IMAC on HisTrap HP (GE Healthcare) as described [20 ]. The eluted fractions were pooled and concentrated by Amicon Ultra (Millipore) followed by size exclusion chromatography (SEC) on a HiLoad 26/600 Superdex200 column (GE Healthcare). A final chromatography step of the samples on either ResourceQ or Superdex75 10/300 GL (both GE Healthcare) columns was performed when needed. The monomeric scTCR wt380 and scTCR s380 molecules or the heterodimeric dsTCR and cFab molecules were pooled and concentrated as before. SEC was performed using PBS with 300 mM NaCl and protein concentration was determined using the MW and extinction coefficient of each individual protein (Denovix DS-11+ Spectrophotometer). Analytical gelfiltration was performed using Superdex 200 Increase 3.2/300 column (GE Healthcare) run in PBS with 300 mM NaCl.
5
Purification of Sulfolobus clsN proteins
6
Purification of PriSLX and PriX Complexes
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PriSLX complex was expressed and purified essentially as previously described (3 (link)). Cells expressing PriSL were lysed in 10 mM Hepes at pH 7.5, 150 mM NaCl, 14 mM β-mercaptoethanol, and EDTA-free protease inhibitor mini tablets (Thermo Fisher Scientific), using French press. The lysate was centrifuged at 34, 957 × g for 30 min, and the supernatant was heat-treated at 75 °C for 20 min before further centrifugation. The supernatant was purified over a HiTrip Heparin column (GE Healthcare), and the protein was eluted with a NaCl gradient from 150 to 1,000 mM in 10 mM Hepes at pH 7.5 buffer. The protein was further purified over a HiLoad 26/600 Superdex 200 column (GE Healthcare) in 10 mM Hepes at pH 7.5, 150 mM NaCl.
Cells expressing PriX were lysed in 10 mM Hepes at pH 7.5, 100 mM NaCl, and 1 mM DTT, using French press. The lysate was centrifuged at 34, 957 × g for 30 min, and the supernatant was heat-treated at 75 °C for 20 min before further centrifugation. The supernatant was purified over a HiTrip Heparin column, and the protein was eluted with a NaCl gradient from 150 mM to 1,000 mM in 10 mM Hepes at pH 7.5 buffer. The protein was further purified over a HiLoad 26/600 Superdex 200 column in 10 mM Hepes at pH 7.5, 150 mM NaCl.
Cells expressing PriX were lysed in 10 mM Hepes at pH 7.5, 100 mM NaCl, and 1 mM DTT, using French press. The lysate was centrifuged at 34, 957 × g for 30 min, and the supernatant was heat-treated at 75 °C for 20 min before further centrifugation. The supernatant was purified over a HiTrip Heparin column, and the protein was eluted with a NaCl gradient from 150 mM to 1,000 mM in 10 mM Hepes at pH 7.5 buffer. The protein was further purified over a HiLoad 26/600 Superdex 200 column in 10 mM Hepes at pH 7.5, 150 mM NaCl.
7
Expression and Purification of GST-Tagged Proteins
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GST-tagged mDia2 and FMNL2/3 were expressed in E. coli host Rosetta 2 (Sigma, St. Louis, MO, USA) by induction with 1 mM isopropyl-β-D-thiogalactoside (Carl Roth, Karlsruhe, Germany) at 24 °C for 12 h. The bacteria were harvested and lysed by ultrasonication in lysis buffer containing PBS, pH 7.4 supplemented with 2 mM DTT, 1 mM EDTA, 5 mM benzamidine (Carl Roth), 0.1 mM AEBSF (AppliChem, Darmstadt, Germany), Benzonase (1:1000, Merck, Darmstadt, Germany) and 5% (v/v) glycerol. The proteins were then purified from bacterial extracts by affinity chromatography using glutathione-conjugated agarose (Macherey-Nagel, Düren, Germany) and eluted from the column with 20 mM reduced glutathione (Carl Roth) in PBS supplemented with 2 mM DTT, 1 mM EDTA, 5 mM benzamidine, 0.1 mM AEBSF and 5% (v/v) glycerol using standard procedures. The GST tag was cleaved off by PreScission protease (GE Healthcare), and the GST tag was then absorbed on fresh glutathione-conjugated agarose. The proteins in the flow through were further purified by size-exclusion chromatography (SEC) using a HiLoad 26/600 Superdex 200 column (GE Healthcare) controlled by an Äkta Purifier System. The fractions containing mDia2, FMNL2 and −3 were pooled and dialyzed against immunization buffer (150 mM NaCl, 25 mM Tris/HCl, pH 8.0) for generation of polyclonal antibodies.
8
Purification of BtuG2 and BtuG3 Proteins
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The coding regions for the mature parts of bt1954 (btuG2) and bt2095 (btuG3) were cloned into pET28 using NcoI and XhoI. The protein was expressed in BL21(DE3) in LB broth (Lennox L. Broth, Melford) 2.5 h at 37 °C with the addition of 1 mM IPTG (Melford) at OD ~ 0.6 to induce protein expression. Cells were collected by centrifugation, resuspended in TBS (10 mM Tris (Sigma-Aldrich)), 300 mM NaCl; pH 8) lysed at a pressure of 20–23 kpsi using a cell disrupter (Constant Systems 0.75 kW), and purified by nickel affinity chromatography. Protein concentrations were measured using the BCA assay, and when needed, adenosylcobalamin, cyanocobalamin or cobinamide (all from Sigma-Aldrich) was added to a molar ratio 1:2 protein:corrinoid. The samples were incubated at 4 °C for 30 minutes and subjected to SEC using a HiLoad 26/600 Superdex 200 column (GE healthcare).
9
Monoclonal Antibody REGN10933 Production
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Publicly available sequences of variable domains of the monoclonal antibody REGN10933 were used to synthetize the corresponding cDNA fragments and cloned into a proprietary expression vector at Evitria AG (Schlieren, Switzerland). Generated vectors containing the constant immunoglobulin chains were used for transfection in Chinese hamster ovary cells by Evitria. Sterile filtered cell supernatants were purified via affinity purification with a HiTrap MabSelect column (Cytiva) followed by size exclusion chromatography using a HiLoad 26/600 Superdex 200 column (GE Healthcare, Danderyd, Sweden) in PBS, pH 7.4. Selected fractions were pooled and quality controlled by Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE), size exclusion chromatography and endotoxin measurement before use in assays.
10
Purification and Complex Formation of PPARα-LBD
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The human PPARα–LBD (NM_001001928, residues 196–468) was expressed as N-terminal 6×His fusion protein from the expression vector pET24a (Novagen, Darmstadt, Germany). The BL21 (DE3) cells transformed by expression plasmids were grown in LB broth at 25 °C to an OD600 of ~1.0, and induced with 0.1 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) at 16 °C for 14–16 h. Cells were harvested and sonicated in 200 mL of extraction buffer (20 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, and 25 mM imidazole) per 6 L of cells. The lysate was centrifuged at 20,000 rpm for 30 min, and the supernatant was loaded on a 5 mL Ni-loaded HiTrap HP column (GE Healthcare). The column was washed with extraction buffer, and the protein was eluted with a gradient of 25–500 mM imidazole. The PPARα–LBD was further purified by gel filtration using a HiLoad 26/600 Superdex 200 column (GE Healthcare, Pittsburgh, PA, USA) (elution buffer, 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM DTT). To prepare the protein–ligand complex, we added a five-fold molar excess of sanguinarine to the purified protein, followed by a filter concentration to 10 mg/mL. The PPARα–LBD was complexed with two-fold molar of a SRC1-2 peptide (RHKILHRLLQEGSP) before filter concentration.
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