The paired antibody VH/VL chains were cloned into Igγ and Ig expression vectors using T4 ligase (NEB). Antibodies produced from cell culture supernatants were purified immediately by affinity chromatography using recombinant Protein G-Agarose (Thermo Scientific) according to the manufacturer’s instructions to purify IgG. The purified antibodies were concentrated by an Amicon ultracentrifuge filter device (molecular weight cut-off 10 kDa; Millipore) to a volume of 0.2 mL in PBS (Life Technologies), and then stored at 4 °C or −80 °C for further characterization.
Amicon ultracentrifuge filter device
Manufactured by Merck Group
The Amicon ultracentrifuge filter device is a laboratory equipment used for the separation and concentration of macromolecules, such as proteins, enzymes, and nucleic acids, from complex solutions. The device utilizes a semi-permeable membrane to selectively retain the desired macromolecules while allowing smaller molecules and solvents to pass through, enabling the concentration and purification of the target analytes.
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Lab products found in correlation
Recombinant protein g agarose, by Thermo Fisher Scientific (4 mentions)
T4 ligase, by New England Biolabs (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Protease lys c, by Roche (1 mentions)
Expi293f expression system, by Thermo Fisher Scientific (1 mentions)
Nebuilder hifi dna assembly cloning kit, by New England Biolabs (1 mentions)
Expi293 expression system, by Thermo Fisher Scientific (1 mentions)
5 protocols using amicon ultracentrifuge filter device
1
Recombinant Antibody Purification Protocol
2
Antibody Production and Purification
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Antibodies were produced and purified as previously described [21 (link)]. The paired antibody VH/VL chains were cloned into Igγ and Igk expression vectors using T4 ligase (NEB). Antibodies produced from cell culture supernatants were purified immediately by affinity chromatography using recombinant Protein G-Agarose (Thermo Scientific) according to the manufacturer’s instructions to purify IgG. The purified antibodies were concentrated by an Amicon ultracentrifuge filter device (molecular weight cut-off 10 kDa; Millipore) to a volume of 0.2 mL in PBS (Life Technologies), and then stored at 4°C or −80°C for further characterization.
3
Recombinant Human Neutralizing Antibody Production
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The antibody VH/VL and constant region genes were amplified and cloned into the expression vectors AbvecIgG and AbvecIgKappa using the NEBuilder® HiFi DNA Assembly Cloning Kit (NEB). The plasmids of paired IgH and IgL genes were cotransfected into the Expi293F expression system (Thermo Fisher Scientific) following the manufacturer’s protocol to produce recombinant HuNAbs. Antibodies from cell culture supernatants were purified immediately by affinity chromatography using Recombinant Protein G Agarose (Thermo Fisher Scientific) according to the manufacturer’s instructions. The purified HuNAbs were concentrated by an Amicon ultracentrifuge filter device (molecular weight cutoff, 50 kDa; Millipore) to a volume of 0.2 mL in PBS and stored at −80°C. To produce Fab fragments, antibodies were cleaved using Protease Lys-C (Roche) with an IgG to Lys-C ratio of 4000:1 (w/w) in 10mM EDTA, 100mM Tris-HCl, pH 8.5 at 37°C for approximately 12 h. Fc fragments were removed using Protein A Sepharose (GeneScript).
4
Preparation of SWNT-ssDNA Complexes
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SWNTs were obtained from Rice University’s Richard E. Smalley Institute for Nanoscale Science and Technology, which is no longer producing SWNTs. Exactly equivalent material can now be purchased from Sigma-Aldrich [carbon nanotube, single-walled (6,5) chirality, ≥95% carbon basis (≥99% as carbon nanotubes), 0.78-nm average diameter, product number 773735].
One milligram of HiPco SWNTs (batch number 189.2, Rice University) and 2 mg of d(T)30 oligonucleotides (Invitrogen) were added to 2 ml of deionized water in a glass scintillation vial. The vial was placed on ice and sonicated (Vibra-Cell, VC-50; Sonics & Materials) at a power of 10 W at 20 kHz for 90 min using a 2-mm-diameter microprobe tip. After sonication, the sample was centrifuged at 16,000g for 90 min. The supernatant was carefully collected and filtered using a 4-ml Millipore Amicon ultracentrifuge filter device (molecular weight cutoff, 100 kDa). The SWNT-ssDNA (single-stranded DNA) stock solution was stored at 4°C.
One milligram of HiPco SWNTs (batch number 189.2, Rice University) and 2 mg of d(T)30 oligonucleotides (Invitrogen) were added to 2 ml of deionized water in a glass scintillation vial. The vial was placed on ice and sonicated (Vibra-Cell, VC-50; Sonics & Materials) at a power of 10 W at 20 kHz for 90 min using a 2-mm-diameter microprobe tip. After sonication, the sample was centrifuged at 16,000g for 90 min. The supernatant was carefully collected and filtered using a 4-ml Millipore Amicon ultracentrifuge filter device (molecular weight cutoff, 100 kDa). The SWNT-ssDNA (single-stranded DNA) stock solution was stored at 4°C.
5
Recombinant Monoclonal Antibody Production
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The paired antibody VH/VL chains were cloned into Igγ, Igα1 or Igα2 and Ig expression vectors using T4 ligase (NEB). For production of IgG and monomeric IgA, the plasmids with paired heavy chain (IgG, IgA1, IgA2) and light chain genes were co-transfected into Expi293TM expression system (Thermo Fisher Scientific) following the manufacturer’s protocol to produce recombinant monoclonal antibodies. For dIgA antibody production, plasmids of paired heavy chain (IgA1, IgA2) and kappa light chain together with a J chain were co-transfected into Expi293TM expression system (Thermo Fisher Scientific) at the ratio of 1:1:1 following the manufacturer’s instructions. Antibodies produced from cell culture supernatants were purified immediately by affinity chromatography using recombinant Protein G-Agarose (Thermo Fisher Scientific) or CaptureSelectTM IgA Affinity Matrix (Thermo Fisher Scientific) according to the manufacturer’s instructions, to purify IgG and IgA, respectively. The purified antibodies were concentrated by an Amicon ultracentrifuge filter device (molecular weight cut-off 10 kDa; Millipore) to a volume of 0.2 ml in PBS (Life Technologies) (Table S2), and then stored at 4 °C or −80 °C for further characterization.
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