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Amicon ultra 4 50 kda centrifugal filter units

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The Amicon Ultra-4 50-kDa centrifugal filter units are a laboratory equipment product designed for the separation and concentration of macromolecules, such as proteins, from complex solutions. The device utilizes a centrifugal force to pass the sample through a semi-permeable membrane, allowing smaller molecules to pass through while retaining the desired larger molecules.

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Lab products found in correlation

50 ml mini bioreactor tubes, by Corning (4 mentions) Pts201f cartridge, by Charles River Laboratories (4 mentions) Endosafe nexgen mcs instrument, by Charles River Laboratories (4 mentions) Hitrap mabselect sure column, by Cytiva (3 mentions) Rapid high throughput cdna synthesis platform, by Twist Bioscience (2 mentions) Hitrap protein a column, by GE Healthcare (2 mentions) Hitrap mab select xtra, by GE Healthcare (2 mentions) Gibco expicho expression system, by Thermo Fisher Scientific (2 mentions) Akta pure chromatographer, by GE Healthcare (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Mabselect sure resin, by Cytiva (1 mentions) Zeba spin desalting plates, by Thermo Fisher Scientific (1 mentions) Hitrap mabselect sure, by Cytiva (1 mentions)

7 protocols using amicon ultra 4 50 kda centrifugal filter units

1

Scalable Monoclonal Antibody Production

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Sequences of mAbs that had been synthesized (Twist Bioscience) and cloned into an IgG1 monocistronic expression vector (designated as pTwist-mCis_G1) were used for mammalian cell culture mAb secretion. This vector contains an enhanced 2A sequence and GSG linker that allows simultaneous expression of mAb heavy and light chain genes from a single construct upon transfection53 (link). We previously described microscale expression of mAbs in 1 mL ExpiCHO cultures in 96-well plates5 (link). For larger scale mAb expression, we performed transfection (1 to 300 mL per antibody) of CHO cell cultures using the Gibco™ ExpiCHO™ Expression System and protocol for 50 mL mini bioreactor tubes (Corning) as described by the vendor. Culture supernatants were purified using HiTrap MabSelect SuRe (Cytiva, formerly GE Healthcare Life Sciences) on a 24-column parallel protein chromatography system (Protein BioSolutions). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon® Ultra-4 50KDa Centrifugal Filter Units (Millipore Sigma) and stored at 4°C until use. Purified mAbs were tested routinely for endotoxin levels that found to be <30 EU/mg IgG for mouse studies and <1 EU/mg IgG for NHP studies. Endotoxin testing was performed using the PTS201F cartridge (Charles River), with sensitivity range from 10 to 0.1 EU/mL, and an Endosafe Nexgen-MCS instrument (Charles River).
Zost S.J., Gilchuk P., Case J.B., Binshtein E., Chen R.E., Nkolola J.P., Schäfer A., Reidy J.X., Trivette A., Nargi R.S., Sutton R.E., Suryadevara N., Martinez D.R., Williamson L.E., Chen E.C., Jones T., Day S., Myers L., Hassan A.O., Kafai N.M., Winkler E.S., Fox J.M., Shrihari S., Mueller B.K., Meiler J., Chandrashekar A., Mercado N.B., Steinhardt J.J., Ren K., Loo Y.M., Kallewaard N.L., McCune B.T., Keeler S.P., Holtzman M.J., Barouch D.H., Gralinski L.E., Baric R.S., Thackray L.B., Diamond M.S., Carnahan R.H, & Crowe JE J.r. (2020). Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature, 584(7821), 443-449.
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2

Parallel Production of Recombinant mAbs

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For parallel production of recombinant mAbs, we used approaches designated as ‘micro-scale’ or ‘midi-scale’ (26 (link)). For ‘micro-scale’ mAbs expression, we performed transfection (~1 mL per antibody) of CHO cell cultures using a protocol for deep 96-well blocks (Thermo Fisher Scientific), as we previously described (26 (link)). For high-throughput micro-scale mAb purification, clarified culture supernatants were incubated with MabSelect SuRe resin (Cytiva), washed with PBS, eluted, buffer-exchanged into PBS using Zeba Spin Desalting Plates (Thermo Fisher Scientific) and stored at 4°C until use. For ‘midi-scale’ mAbs expression, we performed transfection (~35 mL per antibody) of CHO cell cultures as described by the vendor. MAbs were purified form culture supernatants using HiTrap MabSelect SuRe columns (Cytiva). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon Ultra-4 50 KDa Centrifugal Filter Units (Millipore Sigma) and stored at 4°C until use. To quantify purified mAbs, absorption at 280 nm (A280) was measured using a NanoDrop (Thermo Fisher Scientific).
Gilchuk P., Guthals A., Bonissone S.R., Shaw J.B., Ilinykh P.A., Huang K., Bombardi R.G., Liang J., Grinyo A., Davidson E., Chen E.C., Gunn B.M., Alter G., Saphire E.O., Doranz B.J., Bukreyev A., Zeitlin L., Castellana N, & Crowe JE J.r. (2021). Proteo-Genomic Analysis Identifies Two Major Sites of Vulnerability on Ebolavirus Glycoprotein for Neutralizing Antibodies in Convalescent Human Plasma. Frontiers in Immunology, 12, 706757.
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3

High-Throughput Mammalian mAb Production

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The most somatically mutated representatives from each clonal family and members of all public clonotypes were synthesized using a rapid high-throughput cDNA synthesis platform (Twist Bioscience) and subsequently cloned into an IgG1 monocistronic expression vector (designated as pTwist-mCis_G1) for mAb secretion from mammalian cell culture. This vector contains an enhanced 2A sequence and GSG linker that allows simultaneous expression of mAb heavy- and light-chain genes from a single construct upon transfection.63 (link) We performed transfections of ExpiCHO cell cultures using the Gibco ExpiCHO Expression System and protocol for 50 mL mini bioreactor tubes (Corning) as described by the vendor. Culture supernatants were purified using HiTrap MabSelect SuRe (Cytiva) on a 24-column parallel protein chromatography system (Protein Biosolutions). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon Ultra-4 50-kDa centrifugal filter units (Millipore Sigma) and stored at 4°C until use.
(1996). Proceedings of the National Academy of Sciences of the United States of America, 93(25), 14993.
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4

Purification and Characterization of COVID-19 Neutralizing Antibodies

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The mAbs studied in this paper (COV2-2196, COV2-2130, S309, S2E12, 2B04, 47D11, REGN10933, REGN10987, LY-CoV555, and LY-CoV016) have been described previously31 (link),36 -42 . COV2-2196 and COV2-2130 mAbs were produced after transient transfection using the Gibco ExpiCHO Expression System (ThermoFisher Scientific) following the manufacturer’s protocol. Culture supernatants were purified using HiTrap MabSelect SuRe columns (Cytiva, formerly GE Healthcare Life Sciences) on an AKTA Pure chromatographer (GE Healthcare Life Sciences). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon Ultra-4 50-kDa centrifugal filter units (Millipore Sigma) and stored at −80 °C until use. Purified mAbs were tested for endotoxin levels (found to be less than 30 EU per mg IgG). Endotoxin testing was performed using the PTS201F cartridge (Charles River), with a sensitivity range from 10 to 0.1 EU per mL, and an Endosafe Nexgen-MCS instrument (Charles River). S309, S2E12, REGN10933, REGN10987, LY-CoV016, and LY-CoV555 mAb proteins were produced in CHOEXPI cells and affinity purified using HiTrap Protein A columns (GE Healthcare, HiTrap mAb select Xtra #28-4082-61). Purified mAbs were suspended into 20 mM histidine, 8% sucrose, pH 6.0. The final products were sterilized by filtration through 0.22μm filters and stored at 4°C.
Chen R.E., Winkler E.S., Case J.B., Aziati I.D., Bricker T.L., Joshi A., Darling T.L., Ying B., Errico J.M., Shrihari S., VanBlargan L.A., Xie X., Gilchuk P., Zost S.J., Droit L., Liu Z., Stumpf S., Wang D., Handley S.A., Stine WB J.r., Shi P.Y., Davis-Gardner M.E., Suthar M.S., Knight M.G., Andino R., Chiu C.Y., Ellebedy A.H., Fremont D.H., Whelan S.P., Crowe JE J.r., Purcell L., Corti D., Boon A.C, & Diamond M.S. (2021). In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains. Nature, 596(7870), 103-108.
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5

Production and Characterization of Neutralizing Antibodies

Cited 1 time
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The mAbs studied in this paper (COV2-2196, COV2-2130, S309, S2E12, 2B04, 47D11, REGN10933, REGN10987, LY-CoV555, and CB6) have been described previously6 (link),8 (link),10 (link),44 ,48 -51 (link). COV2-2196 and COV2-2130 mAbs were produced after transient transfection using the Gibco ExpiCHO Expression System (ThermoFisher Scientific) following the manufacturer’s protocol. Culture supernatants were purified using HiTrap MabSelect SuRe columns (Cytiva, formerly GE Healthcare Life Sciences) on an AKTA Pure chromatographer (GE Healthcare Life Sciences). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon Ultra-4 50-kDa centrifugal filter units (Millipore Sigma) and stored at −80 °C until use. Purified mAbs were tested for endotoxin levels (found to be less than 30 EU per mg IgG). Endotoxin testing was performed using the PTS201F cartridge (Charles River), with a sensitivity range from 10 to 0.1 EU per mL, and an Endosafe Nexgen-MCS instrument (Charles River). S309, S2E12, REGN10933, REGN10987, CB6, and LY-CoV555 mAb proteins were produced in CHOEXPI cells and affinity purified using HiTrap Protein A columns (GE Healthcare, HiTrap mAb select Xtra #28-4082-61). Purified mAbs were suspended into 20 mM histidine, 8% sucrose, pH 6.0. The final products were sterilized by filtration through 0.22μm filters and stored at 4°C.
Diamond M., Chen R., Winkler E., Case J., Aziati I., Bricker T., Joshi A., Darling T., Ying B., Errico J., Shrihari S., VanBlargan L., Xie X., Gilchuk P., Zost S., Droit L., Liu Z., Stumpf S., Wang D., Handley S., Stine W., Shi P.Y., Garcia-Knight M., Andino R., Chiu C., Ellebedy A., Fremont D., Whelan S., Crowe J., Purcell L., Corti D, & Boon A. (2021). In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains. Research Square.
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6

Scalable Monoclonal Antibody Production

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Sequences of mAbs that had been synthesized (Twist Bioscience) and cloned into an IgG1 monocistronic expression vector (designated as pTwist-mCis_G1) were used for mammalian cell culture mAb secretion. This vector contains an enhanced 2A sequence and GSG linker that allows simultaneous expression of mAb heavy and light chain genes from a single construct upon transfection53 (link). We previously described microscale expression of mAbs in 1 mL ExpiCHO cultures in 96-well plates5 (link). For larger scale mAb expression, we performed transfection (1 to 300 mL per antibody) of CHO cell cultures using the Gibco™ ExpiCHO™ Expression System and protocol for 50 mL mini bioreactor tubes (Corning) as described by the vendor. Culture supernatants were purified using HiTrap MabSelect SuRe (Cytiva, formerly GE Healthcare Life Sciences) on a 24-column parallel protein chromatography system (Protein BioSolutions). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon® Ultra-4 50KDa Centrifugal Filter Units (Millipore Sigma) and stored at 4°C until use. Purified mAbs were tested routinely for endotoxin levels that found to be <30 EU/mg IgG for mouse studies and <1 EU/mg IgG for NHP studies. Endotoxin testing was performed using the PTS201F cartridge (Charles River), with sensitivity range from 10 to 0.1 EU/mL, and an Endosafe Nexgen-MCS instrument (Charles River).
Zost S.J., Gilchuk P., Case J.B., Binshtein E., Chen R.E., Nkolola J.P., Schäfer A., Reidy J.X., Trivette A., Nargi R.S., Sutton R.E., Suryadevara N., Martinez D.R., Williamson L.E., Chen E.C., Jones T., Day S., Myers L., Hassan A.O., Kafai N.M., Winkler E.S., Fox J.M., Shrihari S., Mueller B.K., Meiler J., Chandrashekar A., Mercado N.B., Steinhardt J.J., Ren K., Loo Y.M., Kallewaard N.L., McCune B.T., Keeler S.P., Holtzman M.J., Barouch D.H., Gralinski L.E., Baric R.S., Thackray L.B., Diamond M.S., Carnahan R.H, & Crowe JE J.r. (2020). Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature, 584(7821), 443-449.
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7

High-Throughput cDNA Synthesis and Monoclonal Antibody Production

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Sequences of mAbs were synthesized using a rapid high-throughput cDNA synthesis platform (Twist Bioscience) and subsequently cloned into an IgG1 monocistronic expression vector (designated as pTwist-mCis_G1) for mAb secretion from mammalian cell culture. This vector contains an enhanced 2A sequence and GSG linker that allows simultaneous expression of mAb heavy- and light-chain genes from a single construct upon transfection (Chng et al., 2015 (link)). We performed transfections of ExpiCHO cell cultures using the GIBCO ExpiCHO Expression System and protocol for 50mL mini bioreactor tubes (Corning) as described by the vendor. Culture supernatants were purified using HiTrap MabSelect SuRe (Cytiva, formerly GE Healthcare Life Sciences) on a 24-column parallel protein chromatography system (Protein Biosolutions). Purified monoclonal antibodies were buffer exchanged into PBS, concentrated using Amicon Ultra-4 50-kDa centrifugal filter units (Millipore Sigma) and stored at 4°c until use.
Chen E.C., Gilchuk P., Zost S.J., Suryadevara N., Winkler E.S., Cabel C.R., Binshtein E., Chen R.E., Sutton R.E., Rodriguez J., Day S., Myers L., Trivette A., Williams J.K., Davidson E., Li S., Doranz B.J., Campos S.K., Carnahan R.H., Thorne C.A., Diamond M.S, & Crowe JE J.r. (2021). Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals. Cell Reports, 36(8), 109604.
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