The designed fusion proteins covering proMMP activation sequences were expressed using an E. coli BL21 expression system. Following the 0.5 mM IPTG induction at OD600nm = 0.5–0.6, the bacterial culture protein production was facilitated for 3 h at 37 °C, with shaking. Then, the bacteria were spun down, and the pellet was suspended in buffer A (10 mM sodium phosphate, 500 mM NaCl, and 5 mM imidazole, pH 7.4) and sonicated (15 min at 16 °C, pulse 6s, amplitude 70%). Supernatant of the soluble proteins was then loaded onto the HisTrap™ Excel (GE Healthcare, Chicago, IL, USA) column in buffer A and eluted with a linear gradient of 0–100% of 1 M imidazole in buffer A in 20 column volumes (CV). Protein containing fractions were pooled together and exchanged into 50 mM Tris pH 7.5 and then purified by ion exchange chromatography using a MonoQ 4.6/100 PE column (GE Healthcare, Chicago, IL, USA) with a linear gradient of 0–100% 50 mM Tris pH 7.5, 1 M NaCl in 15 CV. Purity of all the products was verified by SDS-PAGE.
Monoq 4.6 100 pe column
Manufactured by GE Healthcare
Sourced in United States, China, Poland
The MonoQ 4.6/100 PE column is a laboratory equipment designed for ion-exchange chromatography. It is a pre-packed column with a strong anion-exchange resin, suitable for the separation and purification of biomolecules such as proteins, peptides, and nucleic acids. The column has a bed volume of 1.7 mL and is compatible with standard liquid chromatography systems.
Automatically generated - may contain errors
Lab products found in correlation
Histrap excel, by GE Healthcare (1 mentions)
Q sepharose, by GE Healthcare (1 mentions)
Sp sepharose, by GE Healthcare (1 mentions)
Hi5 cells, by Thermo Fisher Scientific (1 mentions)
Pfastbac1, by Thermo Fisher Scientific (1 mentions)
Histrap hp 5 ml column, by GE Healthcare (1 mentions)
Bac to bac baculovirus expression system manual, by Thermo Fisher Scientific (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Histrap excel column, by GE Healthcare (1 mentions)
4 protocols using monoq 4.6 100 pe column
1
Purification of Fusion Proteins with MMP Activation
2
Purification and Characterization of Edible Mushroom Compounds
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Fresh B. edulis fruiting bodies were purchased from a vendor of edible mild mushrooms in Yunnan, China and kept at −20 °C. SP-Sepharose, Q-Sepharose, Mono Q 4.6/100 PE column and Superdex 75 10/300 GL prepacked column were obtained from GE Healthcare (China). Sprague-Dawley rats and ICR mice were provided by Xinglong Experimental Animal Breeding Factory in Haidian District, Beijing. Reagents were of the finest grade available.
3
Production and Purification of H5 Hemagglutinin
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The sequence encoding the ectodomain of HA from NC22/17-HA 131Δ or NC22/17-HA 131E was cloned into the baculovirus transfer vector pFastBac1 (Invitrogen), as previously described (60 (link)), in-frame with an N-terminal gp67 signal peptide for secretion, a C-terminal thrombin cleavage site, a trimerization foldon sequence, and a 6× His-tag at the extreme C terminus for purification. Transfection and virus amplification were performed according to the Bac-to-Bac baculovirus expression system manual (Invitrogen). HA protein was produced by infecting suspension cultures of Hi5 cells (Invitrogen) for 2 days. Soluble HA was recovered from the cell supernatants by metal affinity chromatography using a HisTrap HP 5-ml column (GE Healthcare), and purified by ion-exchange chromatography (IEX) using a Mono-Q 4.6/100 PE column (GE Healthcare). As H5 HA consisted of a mixture of uncleaved HA0 and cleaved HA1/HA2, IEX-purified HA was digested with TPCK trypsin (New England Biolabs, 10 mU trypsin/mg HA, overnight at 4°C) to produce uniformly cleaved HA1/HA2, and then purified by gel filtration chromatography using a Superdex-200 10/300 GL column (GE Healthcare) with a running buffer (pH 8.0) of 20 mM Tris-HCl and 50 mM NaCl. The collected protein fractions were concentrated to 10 mg/mL for further use.
4
Purification of Recombinant Proteins from E. coli
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Protein expression was performed in E. coli BL21 cells and was induced by the addition of 0.5 mM isopropyl-β-d -thiogalactopyranoside to the bacterial culture [when at an OD600 (optical density at 600 nm) of 0.5 to 0.6], followed by shaking for 3 hours at 37°C. The bacteria were then spun down, and the pellet was suspended in buffer A [10 mM sodium phosphate, 500 mM NaCl, and 5 mM imidazole (pH 7.4)]. The pellet suspension was then sonicated and spun down. Soluble proteins were purified with a HisTrap Excel column (GE Healthcare, Poland) in buffer A with a linear gradient of 0 to 100% of 1 M imidazole in buffer A in 20 column volumes. Fractions containing the protein of interest were pooled, and the buffer was exchanged to 50 mM tris (pH 7.5). Last, the protein of interest was purified by ion-exchange chromatography with a MonoQ 4.6/100 PE column (GE Healthcare, Poland) with a linear gradient of 0 to 100% of 50 mM tris (pH 7.5) and 1 M NaCl in 15 column volumes.
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