Recombinant plasmids containing S1, S1T1, S1T2 and S1T3 were respectively transformed into E. coli DH10Bac. After cultured for 12–14 h, the positive recombinant bacmids were generated which were screened by blue-white selection and verified by PCR. The recombinant bacmids were then transfected into SF9 cells by using Lipofectamine 2000 (Invitrogen, USA). Viral supernatant from positive wells were collected and stored at -80 °C. For each sample, the positive virus was amplified and the virus titer was determined for the following infection. Finally, the truncated S1 recombinant proteins were expressed in the amplified recombinant baculovirus infected SF9 cells with the MOI of 0.1. The supernatants containing truncated S1 proteins were then separated from cell cultures at 72 h post infection. For purification, 200 mL supernatant was added to Ni–NTA column (Yeasen, China). After washing with 10 mM imidazole, the purified truncated S1 recombinant protein was eluted with 5 mL of 400 mM imidazole and stored at -80 °C.
Ni nta column
Manufactured by Yeasen
Sourced in China
The Ni-NTA column is a chromatography column used for the purification of histidine-tagged proteins. It contains nickel-nitrilotriacetic acid (Ni-NTA) resin, which binds to the histidine tag on the target protein, allowing it to be separated from other components in the sample.
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4 protocols using ni nta column
1
Expression and Purification of SARS-CoV-2 S1 Proteins
2
Recombinant Protein Expression and Purification
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The genes encoding highly immunogenic proteins (HIPs) were amplified from the genome of strain SC19 and cloned individually into linearized pET-28a or pET-32a vector. The generated plasmid was transformed into E. coli BL21 (DE3), and the expression of recombinant protein was induced with 1mM isopropyl-β-D-thiogalactoside at 30°C for 4 hours. The E. coli cells were harvested by centrifugation at 8,000×g for 10 min at 4°C and lysed by high-pressure cell disruptor at 4°C. Cell debris was removed by centrifugation at 34,000×g at 4°C for 22 min, and the supernatant was filtered through 0.22 μm filters. The recombinant proteins were purified using a Ni-NTA column (Yeasen, Wuhan, China) and concentrated by ultrafiltration (Millipore). Protein concentration was determined by SDS-PAGE using BSA as a standard. The levels of endotoxin present in the purified proteins were determined using the Limulus Amebocyte Lysate (LAL) Endotoxin Quantitation Kit (Xiamen Bioendo Technology Co., Ltd, Xiameng China) and were in the range of 0.05-1.50 EU/mL.
3
Recombinant Protein Expression and Purification from E. coli
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E. coli BL21(DE3) was transformed with pET28a-ZHER-βGal and cultured in LB medium supplemented with kanamycin (50 mg/liter). The expression of ZHER-βGal was induced by 1 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 18°C overnight when the cell density reached an optical density at 600 nm of 0.6. The cells were collected, resuspended in lysis buffer containing 50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride (PMSF), and lysed by sonication. The supernatants were collected by centrifugation at 10,000g for 30 min at 4°C and then applied onto a nickel nitrilotriacetic acid (Ni-NTA) column (Yeasen). The expressed proteins were eluted with PBS containing 50 to 250 mM imidazole.
4
Large-Scale Recombinant Protein Expression
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For large scale expression, 10 mL overnight culture was inoculated into 400 mL LB media containing 100 µg/mL Kanamycin and incubated at 37 °C at 220 rpm. When OD600 reached 0.6–0.8, protein expression was induced with the final concentration of 0.5 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG). After incubation at 16 °C for an additional 20 h, bacterial pellet was collected by centrifugation and resuspended in Equilibration buffer (200 mM NaCl, 50 mM Tris-HCl, 10 mM imidazole, pH 8.0) with protease inhibitor cocktail (Transgen, Beijing, China). Bacterial disruption was performed in a high-pressure homogenizer for optimal lysis on ice. Supernatant was collected after centrifugation and filtered through a 0.22 μm filter. After the selective binding of the protein onto a Ni-NTA column (Yeasen, Shanghai, China), elution was achieved by applying a linear gradient of Elution buffer (200 mM NaCl, 50 mM Tris-HCl, 10 mM-500 mM imidazole, pH 8.0). The eluted samples were then evaluated by SDS-PAGE and quantified using the Bradford Assay Kit (Sangon Biotech, Shanghai, China).
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