The recombinant mouse IL9 (rIL9) gene with a C-terminal hexahistidine and ALFA tag was cloned between the BamHI and NotI site in the pVL1393 baculovirus transfer vector. The recombinant baculovirus was generated by cotransfection of the BestBac 2.0v linearized baculovirus genome (Expression Systems) into Sf9 insect cells (Thermo Fisher Scientific). The rIL9 protein was produced by infecting High Five insect cells (Thermo Fisher Scientific) with the recombinant baculovirus. The secreted rIL9 protein was purified by Ni-Sepharose chromatography (GE Healthcare). After cleavage by thrombin to remove the hexahistidine and ALFA tag, the mIL9 protein was further purified by SP-Sepharose (GE Healthcare) cation-exchange chromatography and Superdex-200 gel filtration chromatography (GE Healthcare) equilibrated with a buffer containing 20 mmol/L Tris pH 8.0, 200 mmol/L NaCl, and 0.1 mmol/L phenylmethylsulfonyl fluoride. The protein sample buffer was changed into PBS (Gibco) using PD MiniTrap G-25 desalting columns (Cytiva). Endotoxin was removed from rIL9 using Pierce High-Capacity Endotoxin Removal Resin (Thermo Fisher Scientific), and the protein samples were sterilized by 0.2 μmol/L sterile membrane filtration. The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
Superdex 200 gel filtration chromatography
Manufactured by GE Healthcare
Sourced in United States
Superdex 200 is a gel filtration chromatography medium used for the separation and purification of proteins, protein complexes, and other biomolecules. It is a cross-linked agarose-dextran matrix that provides a porous structure for the separation of molecules based on their size and shape.
Automatically generated - may contain errors
Lab products found in correlation
Ni2 nta affinity chromatography, by GE Healthcare (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sf9 insect cells, by Thermo Fisher Scientific (1 mentions)
Sp sepharose, by GE Healthcare (1 mentions)
Pd minitrap g 25 desalting column, by Cytiva (1 mentions)
Ni sepharose chromatography, by GE Healthcare (1 mentions)
High five insect cells, by Thermo Fisher Scientific (1 mentions)
Pierce high capacity endotoxin removal resin, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Isopropyl 1 thio β d galactopyranoside, by Merck Group (1 mentions)
Hitrap q ff, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Thioflavin t, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Glutathione agarose beads, by GoldBio (1 mentions)
Hitrap q anion exchange chromatography, by GE Healthcare (1 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (1 mentions)
Pet28a vector, by Thermo Fisher Scientific (1 mentions)
5 protocols using superdex 200 gel filtration chromatography
1
Recombinant Mouse IL9 Protein Production
2
Purification of Anti-α-synuclein Antibodies
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Anti-α-synuclein monoclonal antibody was purchased from BD Biosciences (#610787, San Diego, CA, USA). Isopropyl-1-thio-β-D -galactopyranoside (IPTG), glycine, thioflavin T (ThT), proteinase K (PK) and protease inhibitor cocktail were purchased from Sigma-Aldrich (St Louis, MO, USA). Anion-exchange chromatography (HiTrap Q FF, #17-5053-01) and Superdex-200 gel filtration chromatography (#17-5175-01) columns were purchased from GE Healthcare (Fairfield, CT, USA).
3
Purification of Aspergillus fumigatus CafA Protein
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A codon-optimized gene encoding CafA (residues 75-287) of A. fumigatus was cloned between the EcoRI and NotI sites of the pGEX4T3 vector. This vector harbors a thrombin-cleavable N-terminal glutathione S-transferase. The resulting construct was transformed into Escherichia coli BL21 (DE3) cells, grown in Lysogeny Broth medium at 37°C. When the optical density at 600 nm (OD600) reached 0.6-0.7, protein expression was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 16 h at 20°C.
After harvesting cells and lysing using a microfluidizer, CafA protein was purified using glutathione agarose beads (GoldBio, USA). After thorough washing, the protein was eluted by on-column thrombin cleavage in buffer containing 20 mM TRIS-HCl pH 8.0 and 200 mM NaCl. The protein was further purified by HiTrap Q anion exchange chromatography (GE Healthcare, USA) and Superdex 200 gel filtration chromatography (GE Healthcare). All purification steps were performed on ice or at 4°C.
After harvesting cells and lysing using a microfluidizer, CafA protein was purified using glutathione agarose beads (GoldBio, USA). After thorough washing, the protein was eluted by on-column thrombin cleavage in buffer containing 20 mM TRIS-HCl pH 8.0 and 200 mM NaCl. The protein was further purified by HiTrap Q anion exchange chromatography (GE Healthcare, USA) and Superdex 200 gel filtration chromatography (GE Healthcare). All purification steps were performed on ice or at 4°C.
4
Nitrite Reductase Gene Cloning and Purification
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Total RNA was extracted from B. cereus LJ01, and PrimeScript™ first-strand cDNA synthesis kit (Takara, Dalian, China) was used to synthesize cDNA, following the manufacturer's instructions. The full-length coding sequence of the B. cereus LJ01 (LJ01-NiR) nitrite reductase gene was cloned using primers (5′-ATGAGTTATGAAAAAGTATGGGC-3′ and 5′-CTAAGACGCTAT TACTTCTTCTAAC-3′) and submitted to NCBI GenBank (MG839504). The cDNA was used as the template and 5′-CCAGCTAGCATGAGTTATGAAAAAGTATG-3′ and 3′-TGGCTCGAGCTAAGACGCTAT TACTTC-3′ were used as the primers. The LJ01-NiR gene was PCR amplified with the NheI and XhoI sites, without a signal peptide-encoding sequence. The PCR product was connected to the NheI and XhoI sites of the pET-28a (+) vector (Invitrogen, Carlsbad, CA, USA). The resulting plasmid, pET-28a (+)-LJ01-NiR, was then transformed into E. coli DH5α competent cells. The heterogenous expression of recombinant LJ01-NIR in E. coli were purified by Ni2+-NTA affinity chromatography (GE, USA) and Superdex 200 gel filtration chromatography (GE, USA) using a fast protein liquid chromatography system (AKTA Purifier Explorer, Pharmacia, Uppsala, Sweden), referring to Gao's method with slight modification.14 (link)
5
Purification of SspE and Variants
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The plasmids expressing SspE and its variants were transformed into chemically competent E. coli BL21(DE3) cells. Transformants were inoculated into 50 mL of Luria-Bertani (LB) broth and cultured at 37 °C overnight. The overnight culture was diluted 1:100 (v/v) into LB broth and grown to an OD600 of 0.8 followed by protein induction using 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 16~24 h. After cell collection by centrifugation, the cell pellet was resuspended in binding buffer (25 mM Tris-HCl, 300 mM NaCl, and 20 mM imidazole, pH 8.0) and disrupted using a cell homogenizer (JNBIO, Guangzhou, China). Cell debris was removed by centrifugation at 14,000 g for 45 min at 4 °C, and the supernatant was then purified by Ni2+-NTA affinity chromatography (GE Healthcare) and Superdex 200 gel filtration chromatography (GE Healthcare). Peak fractions were combined and concentrated to 10 mg/mL for crystallization.
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