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Amicon ultracel centrifugal filters

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Amicon Ultracel centrifugal filters are lab equipment designed for sample concentration and buffer exchange. They utilize a semipermeable membrane to selectively retain molecules above a specified molecular weight cutoff while allowing smaller molecules to pass through.

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Lab products found in correlation

Ni nta agarose, by Thermo Fisher Scientific (14 mentions) Expi293f cells, by Thermo Fisher Scientific (14 mentions) Expifectamine 293 transfection kit, by Thermo Fisher Scientific (14 mentions) Sars cov 2 b 1.617.2 rbd protein, by Sino Biological (3 mentions) Quikchange lightning, by Agilent Technologies (1 mentions)

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Amicon filters (181 citations) Amicon centrifugal filters (166 citations) Centrifugal filter (122 citations) Amicon Ultracel-3K centrifugal filters (6 citations) Amicon Ultracel 10K centrifugal filters (5 citations) Amicon Ultracel filters (4 citations) Amicon Ultra 0.5-mL Centrifugal Filters Ultracel 3K (3 citations) Amicon Ultra Ultracel 10k and 100k centrifugal filters (2 citations) Amicon Ultra-15 Centrifugal Filters with Ultracel-10K membranes (2 citations) Amicon ultra-15 centrifugal filters ultracel-3 K (2 citations)

14 protocols using amicon ultracel centrifugal filters

1

Production of Recombinant SARS-CoV-2 Proteins

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Recombinant soluble S and RBD proteins from Wuhan-1 and B.1.1.529 SARS-CoV-2 strains were expressed as described (Amanat et al., 2021 (link); Stadlbauer et al., 2020 (link)). Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection of DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants were harvested 3 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).
Ying B., Scheaffer S.M., Whitener B., Liang C.Y., Dmytrenko O., Mackin S., Wu K., Lee D., Avena L.E., Chong Z., Case J.B., Ma L., Kim T., Sein C., Woods A., Berrueta D.M., Carfi A., Elbashir S.M., Edwards D.K., Thackray L.B, & Diamond M.S. (2022). Boosting with Omicron-matched or historical mRNA vaccines increases neutralizing antibody responses and protection against B.1.1.529 infection in mice. bioRxiv.
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2

SARS-CoV-2 Recombinant RBD Protein Production

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Recombinant Wuhan-1 and XBB.1 RBD proteins were a gift of D. Edwards (Moderna). Recombinant BA.5 and BQ.1.1 RBD proteins were produced in Expi293F cells (ThermoFisher) by transfection of DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants were harvested three days post-transfection, recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), and then buffer exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).
Ying B., Darling T.L., Desai P., Liang C.Y., Dmitriev I.P., Soudani N., Bricker T., Kashentseva E.A., Harastani H., Schmidt A.G., Curiel D.T., Boon A.C, & Diamond M.S. (2023). A bivalent ChAd nasal vaccine protects against SARS-CoV-2 BQ.1.1 and XBB.1.5 infection and disease in mice and hamsters. bioRxiv.
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3

Recombinant SARS-CoV-2 Spike Protein Production

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Recombinant soluble S proteins from different SARS-CoV-2 strains were expressed as previously described (Amanat et al., 2021 (link); Stadlbauer et al., 2020 (link)). Briefly, mammalian cell codon-optimized nucleotide sequences coding for the soluble ectodomain of the S protein of SARS-CoV-2 including a C-terminal thrombin cleavage site, T4 foldon trimerization domain, and hexahistidine tag were cloned into mammalian expression vector pCAGGS. The spike protein sequence was modified to remove the polybasic cleavage site (RRAR to A), and two pre-fusion stabilizing proline mutations were introduced (K986P and V987P, wild type Wuhan-Hu-1 numbering). Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection with purified DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants from transfected cells were harvested 3 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).
Ying B., Whitener B., VanBlargan L.A., Hassan A.O., Shrihari S., Liang C.Y., Karl C.E., Mackin S., Chen R.E., Kafai N.M., Wilks S.H., Smith D.J., Carreño J.M., Singh G., Krammer F., Carfi A., Elbashir S., Edwards D.K., Thackray L.B, & Diamond M.S. (2021). Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains. bioRxiv.
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4

Recombinant SARS-CoV-2 RBD Protein Production

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Recombinant Wuhan-1 and XBB.1 RBD proteins were a gift of D. Edwards (Moderna). Recombinant BA.5 and BQ.1.1 RBD proteins were produced in Expi293F cells (Thermo Fisher) by transfection of DNA using the ExpiFectamine 293 Transfection kit (Thermo Fisher). Supernatants were collected 3 d after transfection, and recombinant proteins were purified using Ni-NTA agarose (Thermo Fisher), buffer exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).
Ying B., Darling T.L., Desai P., Liang C.Y., Dmitriev I.P., Soudani N., Bricker T., Kashentseva E.A., Harastani H., Raju S., Liu M., Schmidt A.G., Curiel D.T., Boon A.C, & Diamond M.S. (2024). Mucosal vaccine-induced cross-reactive CD8+ T cells protect against SARS-CoV-2 XBB.1.5 respiratory tract infection. Nature Immunology, 25(3), 537-551.
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5

Expression and Purification of SARS-CoV-2 RBD Protein

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Recombinant receptor binding domain of S (RBD), was expressed as previously described (50 (link), 51 (link)). Briefly, RBD, along with the signal peptide (amino acids 1–14) plus a hexahistidine tag were cloned into mammalian expression vector pCAGGS. RBD mutants were generated in the pCAGGS RBD construct by changing single residues using mutagenesis primers. Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection with purified DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants from transfected cells were harvested 4 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer exchanged into phosphate buffered saline (PBS) and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).
Schmitz A.J., Turner J.S., Liu Z., Aziati I.D., Chen R.E., Joshi A., Bricker T.L., Darling T.L., Adelsberg D.C., Alsoussi W.B., Case J.B., Lei T., Thapa M., Amanat F., O’Halloran J.A., Shi P.Y., Presti R.M., Krammer F., Bajic G., Whelan S.P., Diamond M.S., Boon A.C, & Ellebedy A.H. (2021). A public vaccine-induced human antibody protects against SARS-CoV-2 and emerging variants. bioRxiv.
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6

SARS-CoV-2 Variant Protein Expression

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Recombinant soluble S and RBD proteins from Wuhan-1, BA.1, B.1.351 and B.1.617.2 SARS-CoV-2 strains were expressed as described (Amanat et al., 2021 (link); Stadlbauer et al., 2020 (link)). Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection of DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants were harvested 3 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore). SARS-CoV-2 B.1.617.2 RBD protein was purchased from Sino Biological (Cat. # 40592-V08H90).
Ying B., Scheaffer S.M., Whitener B., Liang C.Y., Dmytrenko O., Mackin S., Wu K., Lee D., Avena L.E., Chong Z., Case J.B., Ma L., Kim T.T., Sein C.E., Woods A., Berrueta D.M., Chang G.Y., Stewart-Jones G., Renzi I., Lai Y.T., Malinowski A., Carfi A., Elbashir S.M., Edwards D.K., Thackray L.B, & Diamond M.S. (2022). Boosting with variant-matched or historical mRNA vaccines protects against Omicron infection in mice. Cell, 185(9), 1572-1587.e11.
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7

Production of Stabilized SARS-CoV-2 Spike Protein

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Recombinant S, was expressed as previously described.39 (link) Briefly, a mammalian cell codon-optimized nucleotide sequence coding for soluble S (GenBank: MN908947.3, amino acids 1-1,213) modified to remove the polybasic cleavage site (RRAR to A), but introducing two stabilizing mutations (K986P and V987P, wild-type numbering) and a C-terminal thrombin cleavage site, T4 foldon trimerization domain, and a 6xHIS tag were cloned into mammalian expression vector pCAGGS.40 (link) Recombinant S was produced in Expi293F cells (ThermoFisher, Cat #A14527) by transfection with purified DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher, Cat #A14524). Supernatants from transfected cells were harvested 4 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoScientific, Cat #88222), then buffer exchanged into phosphate buffered saline (PBS) and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore, UFC903024).
Darling T.L., Ying B., Whitener B., VanBlargan L.A., Bricker T.L., Liang C.Y., Joshi A., Bamunuarachchi G., Seehra K., Schmitz A.J., Halfmann P.J., Kawaoka Y., Elbashir S.M., Edwards D.K., Thackray L.B., Diamond M.S, & Boon A.C. (2022). mRNA-1273 and Ad26.COV2.S vaccines protect against the B.1.621 variant of SARS-CoV-2. Med (New York, N.y.), 3(5), 309-324.e6.
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8

SARS-CoV-2 Recombinant Protein Purification

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Recombinant soluble S and RBD proteins from Wuhan-1 SARS-CoV-2 strains were expressed as described32 (link). Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection of DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants were harvested 3 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer-exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).
Desai P., Karl C.E., Ying B., Liang C.Y., Garcia-Salum T., Santana A.C., Caten F.T., Urban JF J.r., Elbashir S.M., Edwards D.K., Ribeiro S.P., Thackray L.B., Sekaly R.P, & Diamond M.S. (2024). Intestinal helminth infection impairs vaccine-induced T cell responses and protection against SARS-CoV-2. bioRxiv.
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9

Recombinant SARS-CoV-2 RBD Protein Production

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Recombinant RBD proteins from Wuhan-1 and BA.5 SARS-CoV-2 strains were expressed as described 55 (link). Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection of DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants were harvested 3 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).
Chatterjee A, & Singh K.K. (2001). Uracil-DNA glycosylase-deficient yeast exhibit a mitochondrial mutator phenotype. Nucleic Acids Research, 29(24), 4935-4940.
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10

Recombinant SARS-CoV-2 Spike Protein Production

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Recombinant soluble S and RBD proteins from Wuhan-1, BA.1, and BA.5 SARS-CoV-2 strains were expressed as described43 (link),44 (link). Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection of DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants were harvested 3 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore). SARS-CoV-2 B.1.617.2 RBD protein was purchased from Sino Biological (Cat. # 40592-V08H90).
Scheaffer S.M., Lee D., Whitener B., Ying B., Wu K., Jani H., Martin P., Amato N.J., Avena L.E., Berrueta D.M., Schmidt S.D., O’Dell S., Nasir A., Chuang G.Y., Stewart-Jones G., Koup R.A., Doria-Rose N.A., Carfi A., Elbashir S.M., Thackray L.B., Edwards D.K, & Diamond M.S. (2022). Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant. bioRxiv.
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