Coding sequences of mature proteins without signal peptide were amplified with primers listed in Table S2 and cloned into expression vector pETSUMO (Thermo Fisher Scientific, Massachusetts, USA). Correctness of the resulting constructs pETSUMO-ChElp2 and pETSUMO-Vd2LysM were confirmed by DNA sequencing and introduced into E. coli strains BL21 and Origami, respectively. Both proteins were produced at 28°C with 0.2 mM IPTG. Cell culture was pelleted by centrifugation at 4000 g for 40 min at 4°C, and the pellet was resuspended in 20 mL lysis buffer (Table S2), shaken at 4°C for at least two hours and centrifuged at 10,000 g for 1 h. The supernatant was collected and purified using His60 Ni Superflow resin (TaKaRa, California, USA) on a BioLogic LP system (Bio-Rad, California, USA). The resulting protein was dialyzed 3 L of 20 mM Tris, 150 mM NaCl, 5 % glycerol, pH 8.0 while 5 µL of cleavage protein ULP1 was added into the dialysis membrane to cleave-off the 6×His-SUMO tag. Next day, protein solution was collected and subjected to purification using His60 Ni Superflow resin (TaKaRa, California, USA) to remove 6×His-SUMO tag from the protein preparations. Eventually, LysM proteins were dissolved in 20 mM Tris, 150 mM NaCl, 5% glycerol, pH 8.0 and concentrated to a high concentration.
His60 ni superflow resin
Manufactured by Takara Bio
Sourced in United States, China
His60 Ni Superflow Resin is a nickel-based affinity resin used for the purification of histidine-tagged proteins. It features a uniform bead size and high binding capacity to facilitate efficient protein capture and recovery.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 75 16 60, by Cytiva (4 mentions)
Amicon centrifugal filter unit, by Merck Group (4 mentions)
Endo h, by New England Biolabs (3 mentions)
Hiload 16 600 superdex 200 pg column, by GE Healthcare (3 mentions)
Gsh sepharose, by Takara Bio (2 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (2 mentions)
Octyl β d glucopyranoside og, by Merck Group (2 mentions)
Isopropyl β d thiogalactoside iptg, by Americanbio (2 mentions)
Tris 2 carboxyethyl phosphine tcep, by Thermo Fisher Scientific (2 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (2 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phospho l serine, by Avanti Polar Lipids (2 mentions)
Dioleoyl sn glycero 3 phospho l serine, by Avanti Polar Lipids (2 mentions)
L α phosphatidylinositol 4 5 bisphosphate, by Avanti Polar Lipids (2 mentions)
N dodecyl β d maltopyranoside ddm, by Avanti Polar Lipids (2 mentions)
Superdex 200 16 60 column, by GE Healthcare (2 mentions)
Ctb standard, by Merck Group (2 mentions)
Goat anti rabbit igg hrp, by Southern Biotech (2 mentions)
Rabbit anti vp1 polyclonal antibody, by Alpha Diagnostic (2 mentions)
Recombinant sabin 1 vp1, by Alpha Diagnostic (2 mentions)
Rosetta 2 de3 placi, by Merck Group (2 mentions)
48 protocols using his60 ni superflow resin
1
Purification of LysM Proteins
2
Expression and Purification of eOD-GT8 Protein
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eOD-GT8 was expressed in HEK293S GnTI−/− cells. Cells were cultured in suspension and transfected using 500 μg eOD-GT8 plasmid with 293 Free Transfection Reagent (Novagen) in 1 L. After 6 days, cells were centrifuged at 4,500 rpm for 20 min and supernatant was filter-sterilized. A His-tag was utilized for purification by adding His60 Ni-Superflow Resin (Takara, Cat #:636660) in the supernatant at 4°C overnight. Ni Resin was washed with a solution of 150mM NaCl, 20 mM Tris pH 8.0, 20 mM Imidazole pH 7.0 and eluted with a solution of 300 mM NaCl, 50 mM Tris pH 8.0, 250 mM Imidazole pH 7.0. Protein was further purified using SEC as previously described. Complexes of eOD-GT8 and P-p1f1Fab were made by mixing equal molar ratio of both proteins for 1 hour at room temperature. Complexes were then mixed with Endo H (NEB, Cat#: P0702S) for 1hour at 37°C. SEC was used to remove any uncomplexed protein and Endo H. Complexes were concentrated to ~10 mg/mL for crystallization trials.
3
Recombinant Protein Purification Workflow
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Clarified lysate was incubated with His60 Ni Superflow Resin (TaKaRa) for 1 hr at 4˚C. Resin was washed with buffer (300 mM NaCl, 50 mM Tris pH 7.5) supplemented with increasing concentrations of imidazole. Protein was eluted with 300 mM Imidazole. The eluent was then incubated with GSH Sepharose (TaKaRa) for 1 hour before being washed by high salt buffer (500 mM NaCl, 50 mM Tris pH 7.5, 5 10 mM DTT) and then low salt buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1 mM DTT). Resin was incubated with low salt buffer supplemented with PreScission protease o/n at 4˚C to cleave the GST tag. Protein was collected, loaded onto a 6 mL Resource Q column, and eluted in a gradient of buffer Q (50 mM Tris pH 8.5, 1000 mM NaCl). Peak fractions were concentrated (Amicon Centrifugal Filter Units, Merck) followed by size exclusion chromatography (Superdex 75 16/60, Cytiva) in a SEC buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT).
4
Purification and Reconstitution of Membrane Proteins
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Chemicals were from the following sources: Isopropyl-β-D-thiogalactoside (IPTG) (AmericanBio), His60 Ni Superflow Resin (Takara), tris (2-carboxyethyl) phosphine (TCEP) (Thermo), Iodixanol solution (60%, w/v) (Cosmo Bio USA), Octyl β-D-glucopyranoside (OG) (EMD Millipore), n-dodecyl-β-D-maltopyranoside (DDM) (Avanti Polar Lipids). All DNA oligonucleotides were purchased from Integrated DNA Technologies. All lipids were obtained from Avanti Polar Lipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS); L-α-phosphatidylinositol- 4,5-bisphosphate [PI(4,5)P2]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) (NBD-PE); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) (NBD-PS); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE); 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] [DGS-NTA(Ni)]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (MPB-PE).
5
Purification of E2 and E^rns Proteins
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In the purification of E2 protein, the leaves were crushed and incubated for 30 min in an extraction buffer 1 (EB 1) consisting of 50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride (NaCl), 10 mM imidazole, 0.5% (v/v) Triton X-100, 50 mM glycine, 100 mM sodium sulfite, 50 mM ascorbic acid, and 1.5% polyvinylpolypyrrolidone. With the purification of Erns protein, an extraction buffer 2 (EB 2) consisting of 50 mM Tris-Cl (pH 8.5), 300 mM NaCl, 50 mM imidazole, 0.3% (v/v) Triton X-100, 100 mM glycine, 100 mM sodium sulfite, 1 mM phenyl methyl sulfonyl fluoride, and 1.5% polyvinylpolypyrrolidone was used. After centrifugation, the supernatant from EB 1 or EB 2 was mixed with His60 Ni Superflow Resin (Takara, Kusatsu, Japan) for 1 h at 4°C, and the bound proteins were eluted with 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, and 250 mM imidazole for E2 protein or 300 mM imidazole for Erns protein purification. The eluates were adjusted with 50 mM Tris-Cl (pH 7.2) and 150 mM NaCl. The purified proteins were treated with β-mercaptoethanol, assessed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by Western blotting with an anti-His antibody (BioLegend, San Diego, CA, United States), anti-LOM vaccinated serum, and anti-E2 subunit vaccinated serum.
6
Recombinant GRFT Protein Production
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The codon-optimized gene encoding GRFT (GenBank: FJ594069) was synthesized by BGI Genomics in China and cloned into the pET24b(+) vector (Novagen, Germany) to be in frame with a 6×His-tag. Then the plasmid transformed into competent cells BL21 (Takara, China). The protein expression was induced with IPTG at a final concentration of 1 mM at 16℃ for ten hours. The bacteria cells were harvested, resuspended and further lysed with pulsed sonication on ice. GRFT in the cleared lysate was purified by His60 Ni Superflow Resin (Takara, China) according to manufacturer’s guidelines. Finally, the purified protein was analyzed by SDS-PAGE and western blot.
7
Ni-NTA Protein Purification from E. coli
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The plasmids were transformed into the E. coli BL21 DE3 strain. Fresh transformants were washed from an LB (BD Difco-Fisher Scientific) agar plate and used to inoculate a 1-L culture LB supplemented with kanamycin (50 µg/mL). The cells were grown on 37 °C until OD600 reached 0.4 to 0.5 and induced with 0.5 mM IPTG. The cells were harvested after overnight cultivation on 18 °C, 220 rpm. The cells were opened with sonication in binding buffer (BB: 25 mM Hepes, pH 7.5; 300 mM NaCl; 10 mM imidazole; 2 mM CaCl2; 2 mM β-ME). Filtered lysate was incubated with 1 mL previously buffer-equilibrated Ni-beads (His60 Ni Superflow Resin, TaKaRa) for 30 min. Bound protein was washed with BB on a gravity column and eluted with 300 mM imidazole. Fractions were resolved on a 15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and stained with SimplyBlue SafeStain.
8
Expression and Purification of eOD-GT8 Protein
9
Purification of Recombinant Protein
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Clarified lysate was incubated with His60 Ni Superflow Resin (TaKaRa) for 1 hr at 4˚C. Resin was washed with buffer (300 mM NaCl, 50 mM Tris pH 7.5) supplemented with increasing concentrations of imidazole. Protein was eluted with 300 mM Imidazole. The eluent was then incubated with GSH Sepharose (TaKaRa) for 1 hour before being washed by high salt buffer (500 mM NaCl, 50 mM Tris pH 7.5, 5 10 mM DTT) and then low salt buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1 mM DTT). Resin was incubated with low salt buffer supplemented with PreScission protease o/n at 4˚C to cleave the GST tag. Protein was eluted and concentrated (Amicon Centrifugal Filter Units, Merck) followed by size exclusion chromatography (Superdex 75 16/60, Cytiva) in SEC buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT).
10
Protein Purification via Affinity and Size Exclusion
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Clarified lysate was incubated with His60 Ni Superflow Resin (TaKaRa). Resin was washed with a buffer (300 mM NaCl, 50 mM Tris pH 7.5) supplemented with increasing concentrations of imidazole. Protein was eluted with 300 mM Imidazole. Protein was eluted and concentrated (Amicon Centrifugal Filter Units, Merck) followed by size exclusion chromatography (Superdex 75 16/60, Cytiva) in a SEC buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT).
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