4E10 IgG1(κ) was expressed in FreeStyle 293S cells (Invitrogen) as described by Scherer et al. (2010) (link). IgG was purified using rProtein A Sepharose Fast Flow (GE Healthcare) and digested by papain to obtain Fab, which was purified as described by Cardoso et al. (2007) (link) and stored in 20 mM sodium acetate (pH 5.5) at 10 mg/ml.
Rprotein a sepharose fast flow
Manufactured by GE Healthcare
Sourced in United States
RProtein A Sepharose Fast Flow is a chromatography resin designed for the purification of antibodies and other Protein A-binding proteins. It features a high-flow agarose base matrix and Protein A ligand, enabling efficient and rapid purification.
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Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Protein g sepharose 4 fast flow, by GE Healthcare (1 mentions)
Anti flag m2, by Santa Cruz Biotechnology (1 mentions)
Anti myc, by GE Healthcare (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
In fusion advantage pcr cloning kit, by Takara Bio (1 mentions)
Hek293h, by Thermo Fisher Scientific (1 mentions)
Freestyle 293, by Thermo Fisher Scientific (1 mentions)
Anti flag antibody, by Agrisera (1 mentions)
Expi293 cell, by Thermo Fisher Scientific (1 mentions)
Akta purifier fplc, by GE Healthcare (1 mentions)
Hiload 26 600 superdex 200 pg column, by GE Healthcare (1 mentions)
Maxisorp microplate, by Thermo Fisher Scientific (1 mentions)
Ni nta bead, by Qiagen (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Glycine, by Carl Roth (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Cho dg44 cells, by Thermo Fisher Scientific (1 mentions)
Endospecy es 50m kit, by Seikagaku (1 mentions)
12 protocols using rprotein a sepharose fast flow
1
Purification and Digestion of 4E10 IgG1(κ)
2
Immunoprecipitation of Regnase-1 and Act1
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Primary MEFs (WT, Regnase-1–deficient, and Act1 deficient) and HEK293 cells transiently expressing FLAG- or Myc-tagged proteins were used for immunoprecipitation. Cells were disrupted by ultrasonication. After removal of cell debris by centrifugation at 20,000 ×g for 5 min, the cell lysate was incubated with either anti–Regnase-1, anti-Act1 (D-11; Santa Cruz), anti-FLAG M2, or anti-Myc (Sigma) antibodies bound to rProtein A Sepharose Fast Flow, Protein G Sepharose 4 Fast Flow (both GE Healthcare), or Dynabeads Protein G (Thermo Fisher Scientific) for 1 h at 4°C. Beads were then washed twice with Tris buffer. Immunoprecipitated proteins were eluted with 3× SDS sample buffer and subjected to 10% SDS-PAGE (PAGE).
3
Protein Expression and Purification Protocol
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Example 1
Amino acid substitutions were introduced by methods known to those skilled in the art such as using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR, or the In-fusion Advantage PCR cloning kit (TAKARA) to construct expression vectors. Nucleotide sequences of the obtained expression vectors were determined by a method known to those skilled in the art. The produced plasmids were transiently introduced into cells of the human embryonic kidney cancer-derived cell line HEK293H (Invitrogen) or FreeStyle293 (Invitrogen) to express antibodies. From the obtained culture supernatants, antibodies were purified using the rProtein A Sepharose™ Fast Flow (GE Healthcare) by a method known to those skilled in the art. Absorbance at 280 nm of the purified antibody solutions was measured using a spectrophotometer, and antibody concentrations were calculated from the determined values using an absorption coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).
4
Immunoprecipitation of CaTLP1-FLAG in Arabidopsis
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Immunoprecipitation was performed with rProtein A Sepharose Fast Flow (GE Healthcare). Protein extracts were prepared by homogenization of 0.5 g tissue each from wild-type and CaTLP1-FLAG overexpressing Arabidopsis lines in 2 mL buffer containing 50 mM HEPES-KOH, pH 7.5, 0.15 M NaCl, 0.5% (v/v) Triton X-100, and 0.1% (v/v) Tween 20. The homogenates were centrifuged at 10,000 g for 15 min at 4 °C to remove cellular debris. The supernatants were incubated with sepharose beads at 4 °C for 16 h to remove non-specific proteins. Beads were separately incubated with anti-FLAG antibody (Agrisera) at 23 ± 2 °C for 1 h. The pre-extracts were then incubated with anti-FLAG conjugated beads at 4 °C for 16 h, and the beads with immunoaffinity complexes were retrieved. The immunoaffinity complexes were eluted with 50 mL of 2x SDS-sample buffer containing 100 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (w/v) glycerol and 5% (v/v) 2-mercaptoethanol.
5
Antibody Variant Generation and Characterization
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All 117/1,400 antibody variants (WT, Null, and GASDALIE), 10E8.2/iMab and N6-LS-Null used in the humanized mouse experiments were generated in-house as previously described (34 (link)). Briefly, antibody-encoding DNA plasmids were transiently transfected into Expi293 cells (Life Technologies) using a 1:1:1:1 ratio by mass of the heavy-chain and light-chain plasmids encoding either arm of the bispecific antibodies, or a 1:1 ratio by mass of the heavy-chain and light-chain plasmids of N6-LS-Null. IgG was purified from supernatant using rProtein A Sepharose Fast Flow (GE Healthcare). The 117/1,400 variants (WT and Null) and 10E8.4/iMab used in rhesus macaques were produced by Wuxi Biologics, Inc. Size exclusion chromatography was used to assess physicochemical homogeneity of the test antibodies. Fifty μg of the antibodies were analyzed using an AKTA purifier FPLC (GE Healthcare) with column, flow rate, and mobile phase previously described (50 (link)).
6
Purification of Antibody Molecules
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MAbs (different mouse IgG subtypes) were purified from hybridoma supernatants, and the scFv-Fc (human IgG1) fusion protein was purified from CHO supernatant using rProtein A Sepharose Fast Flow (GE Healthcare Life Sciences, 17127901, Chicago, IL, USA). The supernatants were diluted in binding buffer (1.5 M glycine, 3 M NaCl, pH 8.9) at 1:2 ratio. Before loading the samples, the column was equilibrated with 10 column volumes of binding buffer. The antibody solution was applied on the column and subsequently washed with 5 column volumes of binding buffer. Elution was performed with 100 mM glycine, pH 3. The eluates were collected and pH neutralized by adding 1/10 volume of 1M Tris-HCl, pH 8.2. Collected fractions were analyzed by SDS-PAGE and antibody-containing fractions were pooled and dialyzed against PBS. Antibody concentrations were determined with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, ND-2000) by measuring the absorbance at OD280.
7
Recombinant IgG Purification Protocol
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IgG expression vectors were constructed by conjugating the heavy chains to rabbit IgG Fc by using an antibody-expressing positive control vector for IgG expression (ThermoFisher Scientific) as a template. Expi293 cells were cotransfected with expression vectors of heavy and light chains for each IgG, and the supernatant was collected 4 days after transfection. The supernatant was loaded on rProtein A Sepharose Fast Flow (GE Healthcare) equilibrated with PBS. The resin was washed with PBS, and subsequently the IgGs were eluted by 50 mM sodium citrate buffer (pH 3.0). The eluted fractions were immediately neutralized with 2M Tris-HCl (pH 8.0) and further purified by size exclusion chromatography using a HiLoad 26/600 Superdex 200-pg column (GE Healthcare) equilibrated with PBS.
8
Production and Purification of Shiga Toxins
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Purified Stx1 and Stx2 were purchased from Tufts University School of Medicine, Boston, MA, USA. rProtein A Sepharose Fast Flow was bought from GE Healthcare, UK. Ni-NTA beads were purchased from Qiagen (Hilden, Germany). The enzymes used for cloning (NsiI/SalI and EagI/Sall) were bought from Thermo Scientific (MA, USA) and the T4 ligase enzyme was obtained from Invitrogen (CA, USA). HRP/anti-FLAG horseradish peroxidase conjugate and 3’3’-diaminobenzidine (DAB) were acquired from Sigma Aldrich (St Louis, MO, USA). Dulbecco’s medium (DMEM) and fetal bovine serum (FBS) were acquired from GibcoBRL (São Paulo, Brazil). For induction we used IPTG (isopropyl β-D-1-thiogalactopyranoside) from Thermo Scientific. For ELISA assays, we employed 96-wells or 384-well MaxiSorp microplates from Nunc (Rochester, NY, USA); the assays were developed with TMB substrate solution (3,3’,5,5’—tetramethylbenzidine) from Thermo Scientific, and absorbance was determined in a Multiskan EX ELISA reader from Labsystems (Milford, MA, USA).
9
Antibody Purification via Protein A
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MAbs were purified from hybridoma supernatants using affinity chromatography on protein A Sepharose. A chromatography column was packed with rProtein A Sepharose Fast Flow (GE Healthcare Life Sciences, 17127901), equilibrated with washing buffer (1.5 M glycine (Carl Roth, 3908.3), 3 M NaCl (Merck KGaA, 7647-14-5), pH 8.9), and loaded with hybridoma supernatant diluted with washing buffer at 1:3 ratio. Ten column volumes of washing buffer were used to wash the column, followed by elution of bound antibodies with elution buffer (100 mM glycine, pH 3.0). Eluted antibodies were dialyzed against PBS buffer overnight at 4 °C. The purified MAbs were sterile-filtered and stored at 4 °C.
10
Anti-Abrin Polyclonal Antibody Production
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The purified abrin was inactivated by formalin and used to hyperimmunize a rabbit, and 0.5 mL of abrin toxoid (80 mg/mL) was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously to the rabbit. Seven days later, immunization was carried out four times including one booster immunization with the mixture of the abrin toxoid and Freund's incomplete adjuvant as well as three injections with the toxoid at weekly intervals. Ten days after the final injection, the immunized blood was collected by jugular puncture, and the serum was separated for subsequent purification of anti-abrin polyclonal antibodies with rProtein A Sepharose Fast Flow (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The antibody titers were evaluated by enzyme-linked immunosorbent assay (ELISA).
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