Sodium hydroxide (NaOH, 98%) and endotoxin removal spin columns were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Tetrachloroauric(III) acid trihydrate (HAuCl4•3H2O, 99%) was purchased from Alfa Aesar (Haverhill, MA, USA). Tryptone, yeast extract, sodium chloride (NaCl), kanamycin, isopropyl β-D-1-thiogalactopyranoside (IPTG), DNase I, lysozyme, and ampicillin were purchased from BioShop (Burlington, ON, Canada). The proteinase inhibitor and Ni-NTA resin column were purchased from Sigma-Aldrich (St. Louis, MO, USA). Coomassie R-250 dye was purchased from AMRESCO (Solon, OH, USA). Amicon® Ultra centrifugal filters (3K MWCO) were purchased from Merck Millipore (Burlington, MA, USA).
Endotoxin removal spin columns
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Endotoxin removal spin columns are laboratory equipment designed to remove endotoxins from samples. They operate by trapping endotoxins while allowing the desired sample components to pass through the column.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase inhibitor, by Merck Group (1 mentions)
Ni nta resin column, by Merck Group (1 mentions)
Lal chromogenic endotoxin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Printex 90, by Evonik (1 mentions)
Ni nta affinity chromatography, by GE Healthcare (1 mentions)
Biologic lp system, by Bio-Rad (1 mentions)
Protein concentration assay kit, by Sangon (1 mentions)
Ni nta protein purification kit, by Sangon (1 mentions)
Pierce lal chromogenic endotoxin quantitation kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using endotoxin removal spin columns
1
Recombinant Protein Purification
2
Recombinant Mouse S100B Protein Purification
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Recombinant mouse S100B protein was expressed and purified as described previously with minor modifications.23 (link)24 (link) Briefly, the expression vector pET41a-S100B plasmid was transformed into E. coli BL21 (DE3) cells. S100B-expressing BL21 cells were grown in 1 L of LB media with 40 mg/mL kanamycin until the A600 reached 0.9; then 1 mM isopropyl β-D-1-thiogalactopyranoside was added to the bacteria for induction. Cells were harvested and resuspended in lysis buffer (50 mM Tris-HCI, 5 mM EDTA, 1 mM 8 mercaptoethanol, pH 7.5). S100B was purified by HPLC phenyl column (10 μm, 3.9 × 300 mm) according to the manufacturer's instructions, desalted, and freeze dried. Bacterial endotoxin contamination in S100B protein purified from bacterial cultures was minimized with Endotoxin Removal Spin Columns (Thermo Fisher Scientific) according to the manufacturer's instructions. Residual bacterial endotoxin was evaluated using the LAL chromogenic endotoxin quantitation Kit (Thermo Fisher Scientific).
3
Nanoparticle Characterization and Endotoxin Removal
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CNP, 14 nm diameter (Carbon Black, Printex 90, Degussa, Frankfurt, Germany) were characterized and prepared as described earlier [5] (link),[7] (link). Ectoine ((S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, LPS-free, ultrapure 99%, bitop AG, Witten, Germany) and firoin mannosylglycerate, 99% bitop AG, Witten, Germany) were solubilized in PBS. Possible endotoxin contaminations were counteracted by purifying solubilized substances using endotoxin removal spin columns from Thermo Scientific (Germany).
4
Recombinant Mouse IL-33 Protein Purification
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Mouse IL-33 cDNA (Ser109-Ile266) containing a 6X Histidine tags was synthesized with E. Coli favorable codons (Genewise Inc.). The synthesized cDNA was then cloned into pET15b expression vector (Novagen Inc., Madison, WI, USA). The protein was expressed mainly in soluble fraction. Recombinant mouse IL-33 protein was purified by using Ni-NTA affinity chromatography (17-5248-02, GE Healthcare Life Sciences) using BioLogic LP System (Bio-Rad). Following the protein purification, the protein solution was applied through the Endotoxin Removal Spin Columns (#88275, ThermoFisher Scientific) to remove endotoxin. The endotoxin levels in the purified protein was analyzed and confirmed with <0.01 EU per 1 μg of the protein by the LAL method (#88282, ThermoFisher Scientific). The purity of the recombinant proteins was evaluated by Coomassie blue staining analysis and Western blot, which was performed with anti-His antibodies.
5
Recombinant Hsp70 Allergen Purification
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Availability of adequate amounts of the pure allergens is essential to further understand their molecular structures, which is a pre-requisite for the development of more efficacious allergen immunotherapy. The amino acid sequences and coding genes of Hsp70 were retrieved from Uniprot database. The gene of Hsp70 was synthesized and cloned into prokaryotic expression vector pET-28a (+). The expression of His-tagged recombinant Hsp70 in transformant E. coli BL21 was induced by IPTG (final concentration = 1 mmol/L), and the expression products were analyzed by SDS-PAGE. Then, the bacteria were collected and disrupted by sonication. Hsp70 was purified by Ni-NTA protein purification kit (Sangon, C600320-0001), and the recombinant protein was eluted with elution buffer (containing 50mM Tris, 300mM NaCl and 500mM imidazole, Ph 8.0). Then, the endotoxin was removed by Endotoxin Removal Spin Columns (Thermo Fisher, NO. 88273). Purified protein was resuspended in 1×phosphate buffered solution (PBS), and analyzed by SDS-PAGE. The protein concentration was measured by the protein concentration assay kit (Sangon, C503071-0250).
6
Purification of Pyocin S5 from E. coli
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Pyocin S5 was overexpressed from E. coli BL21 (DE3) carrying the plasmid pPyoS5 with initial purification using a cation exchange column (CM16/10 FF, GE Healthcare; buffers: sodium phosphate 50 mM pH 6; sodium phosphate 50 mM pH 6 + NaCl 500mM). Remaining contaminants were removed using an anion exchange column (DEAE 16/10 FF, GE Healthcare; buffers: Tris 20 mM pH 8; Tris 20 mM pH 8 + 500 mM NaCl). The pyocin AP41- ImAP41 complex was purified by nickel affinity chromatography and gel filtration as described previously 23 . For both pyocins, contaminating endotoxins were removed, prior to first storage at -80°C, using endotoxin removal spin columns (Thermo Scientific #88274).
LPS concentration was then verified using the Pierce LAL chromogenic endotoxin quantitation kit (Thermo Scientific 88282). Purified S5 was considered LPS-free when the levels detected were lower than that of the lowest standard of the kit at 0.1EU/mL.
LPS concentration was then verified using the Pierce LAL chromogenic endotoxin quantitation kit (Thermo Scientific 88282). Purified S5 was considered LPS-free when the levels detected were lower than that of the lowest standard of the kit at 0.1EU/mL.
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