Glc-1-P, dTTP, and malachite green reagent were purchased from Sangon Biotech (Shanghai, China). Inorganic pyrophosphatase (YIPP) was purchased from Merck (Germany). D-glucosamine-1-phosphate (GlcNH2-1-P), 2-Azido-2-deoxy-d -glucose-1-phosphate (GlcN3-1-P), glucuronic acid-1-phosphate (GlcA-1-P), and N-acetylglucosamine-1-phosphate (GlcNAc-1-P) were kindly provided by Professor Junqiang Fang from the Shandong University, Qingdao. Ni2+ Sepharose high performance was purchased from GE Healthcare (Uppsala, Sweden). Other chemicals are analytical grade and are commercially available.
Ni2 sepharose high performance
Manufactured by GE Healthcare
Sourced in Sweden
Ni2+ Sepharose high performance is a chromatography resin designed for the purification of histidine-tagged recombinant proteins. It is composed of agarose beads coupled with nickel ions, which selectively bind to the histidine tags often used in protein expression systems. This resin can be used in various protein purification techniques, such as affinity chromatography, to isolate and concentrate the target protein from complex samples.
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Lab products found in correlation
Isopropyl β d thiogalactopyranoside iptg, by Solarbio (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Pbacpak8 transfer vector, by Takara Bio (1 mentions)
Bacpak baculovirus expression system, by Takara Bio (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
4 protocols using ni2 sepharose high performance
1
Enzymatic Synthesis of Sugar Phosphates
2
PLGA Nanoparticle Synthesis and Characterization
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PLGA polymer (the ratio of lactide: glycolide feed was 50:50, Mw: 10 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd., Shandong, China. The Ni2+ Sepharose High Performance was purchased from GE Healthcare Life Sciences (Piscataway, NJ). The isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Solarbio Life Sciences (Beijing, China). The oleic acid-albumin-dextrose-catalase (OADC) enrichment solution was from BD Biosciences (New York, NY, USA), and 7H9 and 7H10 Middlebrook media were from Difco (New York, NY, USA). The mouse monoclonal His-tag antibody was purchased from Proteintech (Wuhan, Hubei, China). Mouse TNF-α, Il-1β, IFN-γ, IgG, and IgA ELISA (enzyme-linked immunosorbent assay) kits were purchased from Neobioscience Technology (Shenzhen, Guangdong, China). The micro-BCA protein assay kit was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA).
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PLGA polymer (the ratio of lactide: glycolide feed was 50:50, Mw: 10 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd., Shandong, China. The Ni2+ Sepharose High Performance was purchased from GE Healthcare Life Sciences (Piscataway, NJ). The isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Solarbio Life Sciences (Beijing, China). The oleic acid-albumin-dextrose-catalase (OADC) enrichment solution was from BD Biosciences (New York, NY, USA), and 7H9 and 7H10 Middlebrook media were from Difco (New York, NY, USA). The mouse monoclonal His-tag antibody was purchased from Proteintech (Wuhan, Hubei, China). Mouse TNF-α, Il-1β, IFN-γ, IgG, and IgA ELISA (enzyme-linked immunosorbent assay) kits were purchased from Neobioscience Technology (Shenzhen, Guangdong, China). The micro-BCA protein assay kit was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA).
3
Enzymatic Synthesis of Glycosidic Compound
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Sugar nucleotide donors UDP-Gal and UDP-Glc were prepared as previously described (Li et al., 2020 (link)). Enzymatic acceptor substrate Glcα-PP-(CH2)11-OPh 1 was synthesized followed the protocol reported previously (Liang et al., 2022 (link)). Ni2+ Sepharose high performance was the product of GE healthcare. Menthol HPLC grade was purchased from Thermo Fisher Scientific. Other chemicals and solvents used were of analytical grade.
4
Recombinant HA Protein Expression
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Recombinant histidine-tagged full-length HA (HA0) originated from IAV H1N1 strain A/Puerto Rico/8/1934 (PR8) was expressed in Sf21 cells using baculovirus as follows. A DNA fragment encoding full-length HA was obtained from cDNA of infected MDCK cells by PCR using specific primers (Supplementary Table. 1 ) and then cloned into a pBacPAK8 transfer vector (Clontech). A mutant construct with an amino-acid substitution of Leu194 to Ala (HA L194A) was prepared using a QuikChange site-directed mutagenesis kit (Agilent). Recombinant baculovirus was generated using the BacPAK Baculovirus Expression System (Clontech). Sf21 cells were infected with each recombinant baculovirus for 3 days and then lysed in lysis buffer (1% NP-40, 50 mm Tris-HCl pH 8.0, 135 mm NaCl, and complete protease inhibitor cocktail (Roche). The supernatants were incubated with Ni2+-sepharose high-performance (GE Life Science) for 16 h at 4 °C. After the beads were washed, each immobilized HA was prepared. For the preparation of soluble HA, immobilized HA was eluted with elution buffer (0.1% NP-40, 20 mm Tris-HCl pH 8.0, 500 mm NaCl, and 1 m imidazole) followed by desalting.
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