FGFRs were prepared as previously described6 (link),34 (link),44 (link),45 (link). Briefly, FGFR1 (residues 458–765), FGFR2 (residues 458–768), FGFR3 (residues 450–758) as well as FGFR4 (residues 445–753), and their mutants, FGFR1C584S, FGFR1V561M, FGFR2V564I, FGFR2V564F and FGFR3V555M, were cloned into a modified pET28a vector in frame with an N-terminal PreScission-cleavable 6×His tag and expressed in E. coli BL21 Rosetta cells. For crystallization, FGFR1C584S was co-expressed with untagged YOPH to induce non-phosphorylated proteins. The harvested cell pellets were lysed in a buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole and 0.5 mM TCEP, and FGFRs were purified over Ni-NTA resin followed by enzymatic digestion with PreScission for 6×His-tag cleavage and further purified by anion exchange chromatography (GE Healthcare). For crystallization, FGFR1C584S was further purified by gel filtration chromatography (GE Healthcare). The proteins were concentrated to 5–16 mg/mL and stored at −80 °C.
Anion exchange chromatography
Manufactured by GE Healthcare
Anion exchange chromatography is a separation technique used to purify and isolate charged molecules, such as proteins, nucleic acids, and other biomolecules. It operates by utilizing the attraction between positively charged functional groups on the stationary phase and negatively charged analytes in the mobile phase. The strength of the interaction can be modulated by adjusting the pH and ionic strength of the buffer, allowing for selective elution and separation of the desired components.
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Lab products found in correlation
Gel filtration chromatography, by GE Healthcare (1 mentions)
Size exclusion chromatography, by GE Healthcare (1 mentions)
Ni agrose affinity resin, by Qiagen (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions)
Glutathione sepharose 4b column, by GE Healthcare (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
4 protocols using anion exchange chromatography
1
FGFR Protein Expression and Purification
2
Purification of Human MTH1 Protein
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The gene of the human mutT homologue MTH1 was ligated into PET 28a vector (Novagen). After the recombinant plasmid was verified by sequencing, it was transformed into E. coli strain BL21 Star (Invitrogen) at 293K, which were grown in LB medium at 37°C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) at 18°C for 16 hours. Bacterial cells were lysed by ultrasonification on ice in buffer containing 100 mM Tris-HCl pH 8.8, 200 mM NaCl, 10% glycerol, 1% TritonX100, 5 mM β-mercaptoethanol. Soluble N-terminally hexa-histidine tagged MTH1 was bound to Ni-agrose affinity resin (Qiagen), washed with a buffer containing 20 mM Tris-HCl pH 8.8, 200 mM NaCl and 10 mM imidazole and eluted with a buffer containing 20 mM Tris-HCl pH 8.8, 250 mM NaCl, and 150 mM imidazole. The eluted protein was concentrated and diluted with a buffer containing 20 mM Tris-HCl pH 8.8, 250 mM NaCl and digested with thrombin for 12–15 h at 277 K. Cut MTH1 was purified by Ni-agrose affinity resin (Qiagen). The protein was further purified with anion exchange chromatography (GE Health), using a linear gradient of 10 mM to 1 M NaCl concentration and size exclusion chromatography (GE Health) at 20 mM Tris-HCl pH 8.8 and 200 mM NaCl [40 (link)].
3
Purification of Recombinant vWbp/Coa Proteins
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E. coli strains containing plasmids pRSET-A-vWbp/Coa or pGEX-5x-1-vWbp/Coa were grown overnight at 37°C in Terrific Broth medium containing 100 μg/ml ampicillin and 35 μg/ml chloramphenicol. The overnight cultures were then diluted 1:50 into fresh Terrific Broth medium, and grown to an OD600 of 0.8–1. Recombinant expression was then induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (Gold Biotechnology, Inc.) for 3 h at 37°C. Bacteria were then harvested, centrifuged and lysed using the French press (SLM Aminco). Soluble recombinant proteins were purified by nickel chelate chromatography (GE Healthcare) and anion exchange chromatography (GE Healthcare) or through a glutathione -Sepharose 4B column (GE Healthcare) according to the manufacturer's manual.
4
FGFR1 C584S Protein Expression and Purification
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Plasmids were expressed in E. coli BL21 Rosetta cells. For FGFR1 C584S, Rosetta cells were co-expressed with YOPH to obtain non-phosphorylated proteins. The colony was inoculated into the liquid LB culture with 50 μg/mL kanamycin at 37 °C and induced at 16 °C for 18 h by the addition of 0.5 mM IPTG between OD600nm of 0.7~0.8. The cells were harvested by 15-min centrifugation at 3000 rpm. The pellets were resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP) and then lysed by a high-pressure homogenizer. The lysate supernatants were obtained after 35 min centrifugation at 18000 rpm and incubated with equilibrated Ni-NTA beads (GE Healthcare) for 1.5 h. Then, the beads were loaded into a gravity flow column and washed with lysis buffer containing 50 mM imidazole. The target proteins were eluted with lysis buffer containing 250 mM imidazole and digested with PreScission protease overnight to remove the N-terminal 6×His tag. Untagged FGFRs were further purified by anion exchange chromatography (GE Healthcare), and peak fractions collected were concentrated to 5–16 mg/mL. For crystallization, the pooled FGFR1 C584S was put through a Superdex 200 column (GE Healthcare) in storage buffer (20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 0.5 mM TCEP).
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