IgG were incubated with IdeS (4 μg of IdeS per 1 mg of IgG) in PBS for 1 hour at 37 °C. The Fc and IdeS A were removed using a mix of Protein A Sepharose® Fast Flow (250 μL per 1 mg digested mAb; GE Healthcare Life Sciences) and Ni Sepharose™ 6 Fast Flow (50 μL per 1 mg digested mAb; GE Healthcare Life Sciences) which were washed twice with PBS before adding to the reaction mixture. After exactly 10 minutes the beads were removed from the F(ab’)2-dilution by filtration in Spin-X tube filters (Costar®) and the filtrate was concentrated in Amicon® Ultra Filters (10k, Millipore). Purified F(ab’)2 fragments were analysed by SDS-PAGE.
Protein a sepharose fast flow
Manufactured by GE Healthcare
Sourced in Germany, Sweden, Japan
Protein A Sepharose Fast Flow is a preparative-scale affinity chromatography medium designed for the purification of immunoglobulins and other Fc-containing proteins. It consists of highly cross-linked agarose beads to which Protein A, a cell wall component of Staphylococcus aureus, is covalently coupled. The medium is characterized by high binding capacity, fast flow properties, and good chemical and physical stability.
Automatically generated - may contain errors
Lab products found in correlation
Freestyle 293 expression system, by Thermo Fisher Scientific (4 mentions)
Ni sepharose 6 fast flow, by GE Healthcare (3 mentions)
Spin x tube filter, by Corning (3 mentions)
Stericup filter unit, by Merck Group (3 mentions)
Pierce igg elution buffer, by Thermo Fisher Scientific (3 mentions)
Amicon ultra filter, by Merck Group (2 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (2 mentions)
Expi293f, by Thermo Fisher Scientific (2 mentions)
Pierce protein a igg binding buffer, by Thermo Fisher Scientific (2 mentions)
Pfuse rigg fc and pfuse2 clig rk2 vectors, by InvivoGen (2 mentions)
Superdex 200 10 300 resin, by GE Healthcare (2 mentions)
Amicon ultra 15, by Merck Group (2 mentions)
Pfuse higg1 fc2 vector, by InvivoGen (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
X tremegene 9 dna transfection reagent, by Roche (1 mentions)
Gammabind g sepharose, by GE Healthcare (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Propidium iodide, by Santa Cruz Biotechnology (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
19 protocols using protein a sepharose fast flow
1
F(ab')2 Generation from Monoclonal Antibodies
2
Soluble Murine and Human Dectin-1 Receptor Production
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The DNA fragment encoding extracellular domain (amino acid residues 73–244) of murine Dectin-1 was cloned using forward primer 5′-aaagatcttacccatacgatgttccagattacgctaattcagggagaaatc-3′ and reverse primer 5′-aaaaatctagacagttccttctcacagatac-3′, and inserted into the BglII sites of pFUSE-hIgG1-Fc2 vector (Invivogen, San Diego, CA), to make HA-tagged murine sDectin-1 (msDectin-1) conjugated with human IgG1 Fc region (Hino et al., 2012 (link)). The DNA fragment encoding extracellular domain (amino acid residues 73–247) of human Dectin-1 was cloned using forward primer 5′-agatcttacccatacgatgttccagattacgctaattcaggaagcaacacattgg-3′ and reverse primer 5′-agatctcattgaaaacttcttctcac-3′ to make HA-tagged human sDectin-1 (hsDectin-1) conjugated with human IgG1 Fc region. HEK293T cells were transfected with pFUSE-hIgG1-Fc2-HA-msDectin-1, pFUSE-hIgG1-Fc2-HA-hsDectin-1, or pFUSE-hIgG1-Fc2 empty vector using X-tremeGENE 9 DNA Transfection Reagent (Roche Applied Science). Then, supernatants were filtrated and gently mixed with Protein A Sepharose Fast Flow (GE Healthcare, Waukesha, WI). The mouse sDectin-1, human sDectin-1, or human IgG1 Fc (control Fc) was eluted with 100 mM Glycine-HCl (pH 3.0), dialyzed with PBS, and concentrated using Amicon Ultra centrifugal filter (Millipore).
3
IgG Digestion and F(ab')2 Purification
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IgG were incubated with IdeS (Dixon, 2014 ) (4 μg of IdeS per 1 mg of IgG) in PBS for 1 h at 37°C. The Fc and IdeS A were removed using a mix of Protein A Sepharose® Fast Flow (250 μL per 1 mg digested mAb; GE Healthcare Life Sciences) and Ni Sepharose™ 6 Fast Flow (50 μL per 1 mg digested mAb; GE Healthcare Life Sciences) which were washed twice with PBS before adding to the reaction mixture. After exactly 10 min the beads were removed from the F(ab’)2-dilution by filtration in Spin-X tube filters (Costar®) and the filtrate was concentrated in Amicon® Ultra Filters (10k, Millipore). Purified F(ab’)2 fragments were analyzed by SDS-PAGE.
4
Purification and Characterization of Broadly Neutralizing Antibodies
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2G12, 4E10 and 2F5 (Polymun Scientific, Vienna, Austria) were procured by the IAVI Neutralizing Antibody Consortium. The recombinant bnMAb IgG1 b12 was expressed by the Center for Antibody Development and Production (The Scripps Research Institute, La Jolla, CA) in Chinese hamster ovary (CHO-K1) cells, purified using affinity chromatography (GammaBind G Sepharose, GE Healthcare) and the purity and integrity was checked by SDS-PAGE. PGT 121–123, PGT 125–131, PGT 135–137, PGT 141–145, PGV04, PDGM1400, PGT151-153 and PG9/16, were transiently expressed with the FreeStyle 293 Expression System (Invitrogen). Antibodies were purified using affinity chromatography (Protein A Sepharose Fast Flow, GE Healthcare) and the purity and integrity was checked by SDS–PAGE.
5
Mammalian Expression of Recombinant Antibodies
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Sequences for heavy and light chains were codon optimized for mammalian expression, synthesized (GenScript), and cloned in the pVRC8400 vector. Equal amounts of heavy and light chain plasmid were co-transfected with Expi293F cells (Thermo Fisher) according to the cell manufacturer’s directions. Following a 6-day incubation, cells were pelleted by centrifugation and supernatant was filtered through 0.22 μm Stericup filter units (EMD Millipore) and then applied to a column containing a 2 mL bed of Protein A Sepharose Fast Flow (GE Healthcare, Chicago, IL) equilibrated with PBS. The column was washed with Protein A IgG Binding Buffer, and antibody was eluted with Pierce IgG Elution Buffer (Thermo Scientific) and pH neutralized with 1 M Tris pH 8 solution. Antibodies were buffer-exchanged to PBS overnight using 10,000 MWCO dialysis cassettes (Thermo Fisher).
6
Monoclonal Antibody Production and Fab Generation
7
Gemcitabine and EGFR Inhibitor Combination Therapy
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Gemcitabine, propidium iodide, anti-RRM1, anti-RRM2 (Santa Cruz Biotechnology, Inc., Dallas, TX); DMEM, RPMI 1649 medium, fetal bovine serum (HyClone Laboratories, Inc., South Logan, UT); human RRM1 and RRM2 produced from HEK293 cells (OriGene Technologies, Inc., Rockville, MD); anti-EGFR, anti-GAPDH, DNA damage antibody sampler kit (Cell Signaling Technology, Boston, MA); C18 Zip-tip, Amicon Ultra-15 centrifugal filters, anti-phospho-Histone H2AX (Ser 139) (α-γ-H2AX) (Merck Millipore, Darmstadt, Germany); protein A-sepharose fast flow (GE Healthcare, Chicago, IL ); afatinib, canertinib, dacomitinib, neratinib, erlotinib (LC Laboratories, Woburn, MA) were purchased from manufacturers indicated in parentheses. Other chemicals were mostly from Sigma-Aldrich Corporation (St. Louis, MO).
8
Purification and Detection of Viral Coat Protein
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Crude extracts from systemically infected N. benthamiana leaves, prepared as previously described [53 (link)], were subjected to centrifugation in 33 mL continuous sucrose gradients (10%–40%) for 2 h at 140,992× g. Aliquots were collected by gravity from the bottom with a capillary tube and analyzed by Western blot anti CP. For CP immunoprecipitation, pools of one of the fractions of 4 independent gradients (4 mL in total) were mixed with an equal volume of 200 mM Tris-HCl, 600 mM NaCl, 10 mM DTT, 0.4% Triton X-100 buffer, pH 7.4 (BI buffer) supplemented with 0.2 % cOmplete EDTA-free Protease Inhibitor Cocktail (Roche, Mannheim, Germany). Then, 10 µL of anti-CP rabbit polyclonal serum was added to the mixture. After five hours of incubation at 4 °C in rotary motion, 50 μL of Protein A Sepharose Fast Flow (GE Healthcare Life Sciences, Uppsala, Sweden) was added, and incubation continued overnight under the same conditions. Immuno-complexes were collected by centrifugation in a tabletop centrifuge and washed three times with BI buffer. Finally, immunoprecipitated CP was eluted from sepharose beads with 80 μL of disruption buffer (125 mM Tris–HCl pH 7.5, 2% SDS, 0.1% bromophenol blue, 6 M urea, 4% glycerol, and 5% β-mercaptoethanol).
9
Transient Expression and Purification of Antibodies
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PGT121, PGT128, PGT135, PG9, PGV04, VRC01, PGT151, and CAP256-VRC26.25 were transiently expressed with the FreeStyle 293 expression system (Thermofisher Scientific). The antibodies were purified using affinity chromatography (Protein A Sepharose Fast Flow; GE Healthcare), and the purity and integrity were checked by SDS-PAGE.
10
Affinity-based Isolation of F(ab')2
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IgG were incubated with IdeS (Dixon, 2014 ) (4 μg of IdeS per 1 mg of IgG) in PBS for 1 hour at 37°C. The Fc and IdeS A were removed using a mix of Protein A Sepharose® Fast Flow (250 μL per 1 mg digested mAb; GE Healthcare Life Sciences) and Ni Sepharose™ 6 Fast Flow (50 μL per 1 mg digested mAb; GE Healthcare Life Sciences) which were washed twice with PBS before adding to the reaction mixture. After exactly 10 minutes the beads were removed from the F(ab’)2-dilution by filtration in Spin-X tube filters (Costar®) and the filtrate was concentrated in Amicon® Ultra Filters (10k, Millipore). Purified F(ab’)2 fragments were analysed by SDS-PAGE.
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