His6-MBP-CbiR was expressed in E. coli Rosetta(DE3) pLysS. Cells were grown to an OD600 of 0.4 at 37°C, and expression was induced with 1 mM IPTG (isopropyl-β-d -thiogalactopyranoside) for 6 h at 30°C. Cells were lysed by sonication in a buffer containing 20 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole (pH 7.4), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mg/ml lysozyme. The protein was purified from the clarified lysate using HisPur nickel-nitrilotriacetic acid (Ni-NTA) resin (Thermo Scientific) and eluted with 250 mM imidazole. Purified protein was dialyzed into a buffer containing 25 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10% glycerol and stored at −80°C.
Hispur nickel nitrilotriacetic acid ni nta resin
Manufactured by Thermo Fisher Scientific
Sourced in United States
HisPur nickel-nitrilotriacetic acid (Ni-NTA) resin is a chromatography resin used for the purification of histidine-tagged recombinant proteins. The resin consists of nickel ions chelated to a nitrilotriacetic acid matrix, which binds to the histidine tags on the target proteins, allowing them to be separated from other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Imidazole, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Precision plus protein all blue prestained protein standard, by Bio-Rad (1 mentions)
Coomassie brilliant blue g 250 protein stain powder, by Bio-Rad (1 mentions)
Sars cov 2 spike neutralizing antibody, by Sino Biological (1 mentions)
Dithiothreitol (dtt), by Avantor (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Sodium phosphate monobasic, by Merck Group (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
3 protocols using hispur nickel nitrilotriacetic acid ni nta resin
1
Purification of His6-MBP-CbiR Protein
2
SARS-CoV-2 Spike Protein Purification
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Ampicillin, isopropyl β-D -thiogalactopyranoside (IPTG), 40-nm gold nanoparticles (AuNPs), sodium phosphate monobasic, sodium chloride, imidazole, sucrose, Tween-20, and Whatman™ Grade 1 qualitative filter paper were purchased from Sigma—Aldrich (St. Louis, MO, USA). Halt protease inhibitor cocktail (100×), 10× phosphate buffered saline (PBS), HisPur nickel-nitrilotriacetic acid (Ni-NTA) resin, and BD Difco Luria-Bertani (LB) Broth were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Coomassie Brilliant Blue G-250 protein stain powder, Precision Plus Protein All Blue Prestained Protein Standards, and Protein Assay Dye Reagent Concentrate were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Bovine serum albumin (BSA) and dithiothreitol (DTT) were purchased from VWR Life Science (Radnor, PA, USA) and LPS Solution (Daejeon, Korea), respectively. A 30% acrylamide-bis solution was purchased from BiOSESANG (Seongnam, Korea). SARS-CoV-2 (2019-nCoV) spike antibodies, chimeric MAb (40150-D006), SARS-CoV-2 spike neutralizing antibody, mouse MAb (40591-MM43), and SARS-CoV spike/S1 protein were purchased from Sino Biological (Wayne, PA, USA). The receptor-binding domain (RBD) of the SARS-CoV-2 spike S1 protein was purchased from HyTest (Turku, Finland). Cultured SARS-CoV-2 was purchased from ATCC (Manassas, VA, USA).
3
Cloning and Expression of M. bovis Genes
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The target genes were amplified from M. bovis strain PD chromosomal DNA. The specific primers for gene point mutations used here in this study are listed in Table S1. The resulting PCR products were digested with the restriction enzymes BamHI and XhoI (New England BioLabs Inc., USA) and then cloned into the pET28a(+) vector using T4 DNA ligase. The resulting recombinant plasmids were separately transformed into Escherichia coli BL21(DE3). Expression of the target proteins was induced by the addition of 1 mM isopropyl-β-d -thiogalactoside (IPTG) for 6 h at 37°C. The recombinant proteins were purified with HisPur nickel-nitrilotriacetic acid (Ni-NTA) resin (Thermo Scientific, USA) and then analyzed via SDS-PAGE and Western blotting with rabbit anti-M. bovis IgG or anti-M. bovis Ab-positive bovine serum. The concentration of each protein was determined with a bicinchoninic acid (BCA) protein assay kit (Cwbiotech, China) before the protein was stored at −20°C for later use.
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