Polyhistidine-tagged B. burgdorferi SpoVG was purified essentially as described previously [1 (link),4 (link)]. Briefly, Escherichia coli Rosetta II (Invitrogen, MA) was transformed with pBLJ132, which consists of spoVG cloned into pET101 [1 (link)]. E. coli were then grown to an OD600 of at least 1.0 in Super Broth (32 g Tryptone, 20 g Yeast Extract, and 5 g NaCl per liter), and recombinant SpoVG expression was induced for 1h by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1mM. Bacteria were harvested by centrifugation at 5400 × g for 30 minutes and frozen at −80°C until needed. Resuspended cells were lysed by sonication with the addition of B-PER bacterial protein extraction reagent to 2% v/v (Thermo-Fisher, MA). Recombinant proteins were purified using MagneHis nickel particles (Promega, WI), then dialyzed against EMSA buffer (50mM Tris-HCl, 25mMKCl, 10% glycerol (v/v), 0.01% Tween 20, 100nM dithiothreitol (DTT), and 1mM phenylmethanesulfonyl fluoride (PMSF)). Proteins were concentrated using 10kDa Amicon centrifugal units (MilliporeSigma, MA) and aliquots were stored at −80°C until needed. Protein purity and concentration were assessed by SDS-PAGE, Quick Start Bradford protein assay (Bio-Rad), and bicinchoninic acid assay (BCA) (Thermo-Fisher, MA).
Amicon centrifugal units
Manufactured by Merck Group
Sourced in United States, United Kingdom
Amicon centrifugal units are laboratory equipment designed for rapid concentration and desalting of macromolecular solutions. These units utilize centrifugal force to separate components of a solution based on their molecular weight. The concentrated sample can then be recovered for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Expi293f cells, by Thermo Fisher Scientific (6 mentions)
Ni nta agarose, by Qiagen (5 mentions)
Ni nta resin, by GoldBio (5 mentions)
Polyethylenimine (pei), by Polysciences (4 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (3 mentions)
Ni nta resin, by Qiagen (3 mentions)
B per bacterial protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Escherichia coli rosetta 2, by Thermo Fisher Scientific (2 mentions)
Magnehis nickel particles, by Promega (2 mentions)
Quick start bradford protein assay, by Bio-Rad (2 mentions)
Ni nitrilotriacetic acid nta agarose, by Qiagen (2 mentions)
Recombinant gamma 10795 cv 100 and delta 10878 cv 100 spike proteins, by R&D Systems (2 mentions)
Expifectamine transfection kit, by Thermo Fisher Scientific (2 mentions)
Expifectamine, by Thermo Fisher Scientific (2 mentions)
Expicho s cells, by Thermo Fisher Scientific (2 mentions)
Ni nta resin, by Abcam (2 mentions)
Pcr primers, by Merck Group (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Biosynth (2 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions)
20 protocols using amicon centrifugal units
1
Purification of Polyhistidine-tagged B. burgdorferi SpoVG
2
Purification of Polyhistidine-tagged B. burgdorferi SpoVG
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Polyhistidine-tagged B. burgdorferi SpoVG was purified essentially as described previously [1 (link),4 (link)]. Briefly, Escherichia coli Rosetta II (Invitrogen, MA) was transformed with pBLJ132, which consists of spoVG cloned into pET101 [1 (link)]. E. coli were then grown to an OD600 of at least 1.0 in Super Broth (32 g Tryptone, 20 g Yeast Extract, and 5 g NaCl per liter), and recombinant SpoVG expression was induced for 1h by adding isopropyl-β-d -thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Bacteria were harvested by centrifugation at 5400×g for 30 min and frozen at −80 °C until needed. Resuspended cells were lysed by sonication with the addition of B-PER bacterial protein extraction reagent to 2% v/v (Thermo-Fisher, MA). Recombinant proteins were purified using MagneHis nickel particles (Promega, WI), then dialyzed against EMSA buffer (50 mM Tris-HCl, 25mMKCl, 10% glycerol (v/v), 0.01% Tween 20, 100 nM dithiothreitol (DTT), and 1 mM phenylmethanesulfonyl fluoride (PMSF)). Proteins were concentrated using 10 kDa Amicon centrifugal units (MilliporeSigma, MA) and aliquots were stored at −80 °C until needed. Protein purity and concentration were assessed by SDS-PAGE, Quick Start Bradford protein assay (Bio-Rad), and bicinchoninic acid assay (BCA) (Thermo-Fisher, MA).
3
Recombinant SARS-CoV-2 Spike Protein Production
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Plasmids encoding mammalian cell codon optimized sequences for the receptor binding domain (RBD) and full-length spike of SARS-CoV-2 was generously gifted from the lab of Dr. Florian Krammer (Amanat et al., 2020 (link))(Icahn School of Medicine, NY, United States). Proteins were produced in Expi293F cells (ThermoFisher Scientific Waltham, MA, United States) according to the manufacturers’ instructions and purified as previously described (Stadlbauer et al., 2021 (link)). Briefly, when culture viability reached 40%, supernatants were collected and spun at 500 x g for 5 minutes. The supernatant was then incubated by shaking overnight at 4°C with 1 mL of Ni-NTA agarose (Qiagen, Germantown MD, United States) per 25 mL of transfected cell supernatant. The following day 10 ml polypropylene gravity flow columns (Qiagen, Germantown, MD, United States) were used to elute the protein. Recombinant RBD was concentrated in a 10 kDa Amicon centrifugal units (Millipore Sigma, Etobicoke, ON, Canada), and recombinant Spike was concentrated in a 50kDa Amicon centrifugal unit (Millipore Sigma, Etobicoke, ON, Canada) prior to being resuspended in phosphate buffered saline (PBS).
4
Concentration and Detection of Waterborne Viruses
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According to the faecal enterococci colony counts determined previously, a total of either 1-L or 20-L water samples were collected in triplicate in autoclaved polypropylene bottles, and were then transported to the laboratory at 4°C for virus concentration (S2 Table ). Next, the samples were processed following standard procedures within 6 h of collection as described by [27 ]. The 20-L water samples were concentrated by ultrafiltration with polycarbonate ultra-filters (Hemoflow F80A, Fresenius Medical Care, Waltham, MA, USA), using a perfusion rate of 1700 mL/min, as described by Hill et al. [28 (link)] while adenovirus detection required further concentration (10,000 X), using Amicon centrifugal units (30,000 MWCO; Merck Millipore, Billerica, MA, USA). Then, the samples were concentrated to a 200-mL final volume, and stored in 40-mL aliquots. Afterwards, RNA was extracted from the 100X concentrated sample to evaluate the FRNA bacteriophage genotypic groups present using RT-qPCR. Subsamples were frozen and stored at −70°C [29 (link)], after which the DNA was extracted.
5
Recombinant SARS-CoV-2 Spike Protein Production and Purification
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Recombinant WA1, Beta, Alpha and Omicron spike proteins and recombinant WA1, Beta, Alpha, Gamma, Delta and Omicron RBDs were generated and expressed in Expi293F cells (Life Technologies, Thermo Fisher Scientific) as previously described [25 (link), 26 (link)]. Proteins were then purified after transient transfections with each respective plasmid. Briefly, the mammalian-cell codon-optimized nucleotide sequence of a soluble spike protein (amino acids 1–1,213) lacking the polybasic cleavage site, carrying two stabilizing mutations (K986P and V987P), a signal peptide, and at the C-terminus a thrombin cleavage site, a T4 fold-on trimerization domain, and a hexahistidine tag was cloned into the mammalian expression vector pCAGGS. https://www.beiresources.org/ ) .Protein was purified using gravity flow purification with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Germany) and concentrated and buffer exchanged in Amicon centrifugal units (EMD Millipore, MA, USA). The purified recombinant proteins were analyzed via reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The desired protein folding was confirmed through ELISAs using the Receptor Binding Domain (RBD)-specific monoclonal antibody CR3022 [27 (link)]. Recombinant Gamma (10795-CV-100) and Delta (10878-CV-100) spike proteins were purchased from R&D Systems (R&D Systems, Bio-Techne, MN, USA).
6
Recombinant SARS-CoV-2 RBD Production
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The pCAGGs SARS-CoV2 RBD plasmid (provided by Florian Krammer) was used for recombinant RBD expression as previously described. FreeStyle 293F cells (ThermoFisher, R79007) were transiently transfected with a mixture of plasmid DNA diluted in PBS (0.67 μg total plasmid DNA per ml of culture) and Polyethylenimine (PEI) (Polysciences, Inc., 23966) at a DNA-to-PEI ratio of 1:3. At six days post-transfection, cultures were harvested by centrifugation at 4,000 x g for 20 min, and supernatant was incubated with Ni-NTA resin (Goldbio) for 2 hours at 4° C with gentle stirring. Resin was collected in columns by gravity flow, washed with 16 column volumes of wash buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 20 mM Imidazole) and eluted in 12mL elution buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 250 mM Imidazole). Eluates were concentrated and exchanged into storage buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl) using an Amicon centrifugal units (EMD Millipore). Protein concentration was determined using an extinction coefficient (33350 M−1cm−1), estimated from amino acid sequence by Expasy online ProtParam, and was further analyzed by SDS-PAGE.
7
Production and Purification of SARS-CoV-2 RBD
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The pCAGGS SARS-CoV2 RBD plasmid (a generous gift from Florian Krammer) was used for the expression of recombinant RBD as previously described (Amanat et al., 2020 (link); Stadlbauer et al., 2020 (link)). FreeStyle 293F cells (ThermoFisher Scientific) were transfected with the plasmid DNA diluted in PBS (0.67 μg total plasmid DNA per ml of culture) using polyethylenimine (Polysciences, Inc.) at a DNA-to-PEI ratio of 1:3. At 6 days post-transfection, cultures were harvested by centrifugation at 4,000 x g for 20 min, and supernatant was incubated with Ni-NTA resin (GoldBio) for 2 h at 4°C. Resin was collected in columns by gravity flow, washed with a wash buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 20 mM Imidazole) and eluted with an elution buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 250 mM Imidazole). Eluant was concentrated in Amicon centrifugal units (EMD Millipore) and buffer was exchanged into the storage buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl). Protein was analyzed by SDS-PAGE, aliquoted, and stored at −80°C.
8
Recombinant SARS-CoV-2 Spike Protein Production
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The recombinant S and RBD proteins were produced as previously described7 (link) in Expi293F cells (ThermoFisher) by transfections of purified DNA using an ExpiFectamine Transfection Kit (ThermoFisher). The soluble version of the spike protein included the S protein ectodomain (amino acids 1–1213), a C-terminal thrombin cleavage site, a T4 foldon trimerization domain and a hexahistidine tag. The protein sequence was modified to remove the polybasic cleavage site (RRAR to A) and two stabilizing mutations (K986P and V987P, wild type numbering). The RBD (amino acids 319–541) also contained a hexahistidine tag. Supernatants from transfected cells were harvested on day three post-transfection by centrifugation of the culture at 4000 g for 20 minutes. Supernatant was then incubated with 6 mL Ni-NTA agarose (Qiagen) for one to two hours at room temperature. Next, gravity flow columns were used to collect the Ni-NTA agarose and the protein was eluted. Each protein was concentrated in Amicon centrifugal units (EMD Millipore) and re-suspended in phosphate buffered saline (PBS).
9
Recombinant SARS-CoV-2 Spike Protein Production
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Recombinant WA1, Beta, Alpha, and Omicron spike proteins and recombinant WA1, Beta, Alpha, Gamma, Delta, and Omicron RBDs were generated and expressed in Expi293F cells (Life Technologies, Thermo Fisher Scientific) as previously described (26 (link), 27 (link)). Proteins were then purified after transient transfections with each respective plasmid. Briefly, the mammalian-cell codon-optimized nucleotide sequence of a soluble spike protein (amino acids 1–1,213) lacking the polybasic cleavage site, carrying two stabilizing mutations (K986P and V987P), a signal peptide, and at the C-terminus a thrombin cleavage site, a T4 fold-on trimerization domain, and a hexahistidine tag was cloned into the mammalian expression vector pCAGGS (https://www.beiresources.org/ ). Protein was purified using gravity flow purification with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Germany) and concentrated, and buffer exchanged in Amicon centrifugal units (EMD Millipore, MA, USA). The purified recombinant proteins were analyzed via reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The desired protein folding was confirmed through ELISAs using the Receptor Binding Domain (RBD)-specific monoclonal antibody CR3022 (28 (link)). Recombinant Gamma (10795-CV-100) and Delta (10878-CV-100) spike proteins were purchased from R&D Systems (R&D Systems, Bio-Techne, MN, USA).
10
Expression and Purification of SARS-CoV-2 RBD Protein
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The pCAGGS SARS-CoV2 RBD plasmid (a generous gift from Florian Krammer) was used for the expression of recombinant RBD as previously described (Amanat et al., 2020 ; Stadlbauer et al., 2020 (link)). FreeStyle 293F cells (ThermoFisher Scientific) were transfected with the plasmid DNA diluted in PBS (0.67 μg total plasmid DNA per ml of culture) using polyethylenimine (Polysciences, Inc.) at a DNA-to-PEI ratio of 1:3. At 6 days post-transfection, cultures were harvested by centrifugation at 4,000 × g for 20 min, and supernatant was incubated with Ni-NTA resin (GoldBio) for 2 h at 4°C. Resin was collected in columns by gravity flow, washed with a wash buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 20 mM Imidazole) and eluted with an elution buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 250 mM Imidazole). Eluant was concentrated in Amicon centrifugal units (EMD Millipore) and buffer was exchanged into the storage buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl). Protein was analyzed by SDS-PAGE, aliquoted, and stored at −80°C.
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