For human IgG1 Fc purification from transgenic chicken serum, 4 M ammonium sulfate was added slowly to chicken serum. The mixture was stirred for overnight at 4 °C, and then centrifuged for 30 min at 4 °C at 10,000 g. The pellet was resuspended in equal volume of 1×PBS to the original serum volume. Then, the mixture was dialyzed against 20 mM sodium phosphate buffer (pH 7.2). The sample was loaded onto a Protein A column (GE Healthcare Bio-Sciences, Uppsala, Sweden), and the protein was eluted with 100 ml gradient of 100 mM Citric acid (pH 2.8). The protein was further purified and fractionated by size-exclusion chromatography (SEC) using a HiLoad Superdex 75 Column (GE Healthcare Bio-Sciences) pre-equilibrated with 20 mM Tris-HCl, 175 mM NaCl (pH 7.4). For human IgG1 Fc purification from egg yolk, egg yolk was separated and diluted with 9 volumes of distilled water. The sample was freezed at -20 °C overnight and thawed at room temperature. After thawing, the supernatant fluid was collected and filtered. Then, the sample was loaded onto a Protein A column (GE Healthcare Bio-Sciences), and the protein was eluted with 100 ml gradient of 100 mM Citric acid (pH 2.8).
Protein a column
Manufactured by GE Healthcare
Sourced in United States, Sweden
The Protein A column is a laboratory tool used for the purification of antibodies. It functions by selectively binding to the Fc region of antibodies, allowing for their separation from other proteins in a sample. The column provides a efficient and reliable method for antibody purification, a critical step in many research and biotherapeutic applications.
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Lab products found in correlation
Polyethylenimine (pei), by Polysciences (7 mentions)
Opti mem 1, by Thermo Fisher Scientific (3 mentions)
Glutamax 1 gibco, by Thermo Fisher Scientific (3 mentions)
Sepharose cl 4b beads, by GE Healthcare (3 mentions)
Expifectamine 293 reagent, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Expi293 expression system, by Thermo Fisher Scientific (2 mentions)
Complete freund s adjuvant, by Merck Group (2 mentions)
Papain agarose, by Merck Group (2 mentions)
Papain agarose, by GE Healthcare (2 mentions)
Linear polyethylenimine, by Polysciences (2 mentions)
Deae sepharose ff column, by GE Healthcare (2 mentions)
Superdex 200 5 150 gl, by GE Healthcare (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
T175 flask, by Corning (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Gentamycin, by Merck Group (2 mentions)
Akta express, by GE Healthcare (2 mentions)
Hybridoma cloning supplement, by GE Healthcare (2 mentions)
70 protocols using protein a column
1
Purification of Human IgG1 Fc from Chicken Serum and Egg Yolk
2
Competitive Binding ELISA for WNV Antibodies
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ELISA plates (Greiner high binding) were coated with 100 ng/well of purified MAb 3.67G (62 (link)) and incubated at 4°C overnight in PBS. The MAbs were purified using protein A columns (GE Life Sciences) according to the manufacturer’s instructions. After washing with PBST, the plates were blocked with ELISA blocking buffer and WNVKUN virions in crude C6/36 culture supernatant was added and incubated for 1 h at 37°C. After washing with PBS/T, a saturating concentration of unlabeled MAbs as hybridoma culture fluid was added in triplicate wells, followed by incubation at 37°C. Without removing the unlabeled MAbs, a predetermined nonsaturating concentration of biotinylated MAb 6B6C-1 or BJ-6E6 (prepared using a B-tag biotinylation kit [Sigma-Aldrich] according to the manufacturer’s instructions) was added, and the plates incubated for a further 1 h at 37°C. After a washing step with PBS/T, bound biotinylated MAb was detected with HRP-conjugated streptavidin and incubation at 37°C for 30 min. After a final wash, ABTS substrate solution was added as above.
3
HIV Neutralization Assay Protocol
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Serum samples from HIV infected individuals were initially tested using TZM-bl target cells as described previously, in triplicate at a 1;200 dilution with the six recombinant replication-competent viruses representing 5 different genetic subtypes, VI 191 (subtype A, tier 2), 92BR025 (subtype C, tier 1B), 92UG024 (subtype D, tier 2), CM244 (subtype AE, tier 2), NL4-3 (subtype B, tier 1A), and AC10.029 (subtype B, tier 2) (59 (link)). For rabbit sera neutralization, 1 ml of serum from immunized animals were preadsorbed with 4 × 107 293F cells (the same cells used for VLP production) for 1 h at room temperature. Next, IgGs from preadsorbed immunized animal sera were purified with protein A columns (GE Healthcare, Chicago, IL) and tested with a previously described virus minipanel as indicated above (25 (link)). Purified IgGs were also tested against pseudotyped viruses with 887-SOS-T and 936-SOS-T envelopes in a TZM-bl assay.
4
Antibody Gene Amplification and Expression
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Picked bone marrow-derived plasma cells were immediately frozen on dry ice for subsequent amplification, cloning, expression, and purification of the VH and VL chains, as described previously.11 (link),34 (link) Briefly, the VH and VL genes were produced by reverse-transcriptase PCR using random hexamers followed by two additional nested PCRs. The nested PCRs utilized 5′ V gene-specific primers and 3′ primers that bound to the heavy, kappa, or lambda constant regions and incorporated compatible ends with Gibson assembly subcloning. After cloning the VH and VL genes into the rhesus IgG1 expression vector (InvivoGen), the heavy and light chains of each Ab were transfected using the suspension-adapted Expi293 expression system (Thermo Fisher Scientific). The following day, the reaction was boosted by adding enhancers. The Ab-containing supernatant was harvested after 5 additional days. All Abs were purified using protein A columns (GE Healthcare).
5
Detailed Antibody Production Protocol
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All antibodies used for flow cytometry, immunohistochemistry and immunofluorescence are detailed in Supplementary Table S1 . For in vivo treatment: the anti-GD2 monoclonal antibody 14G2a was produced in house by secreting hybridoma HB9118 (kindly provided by Prof. Holger Lode, University of Greifswald); the anti-4-1BB (LOB12.3) Rat IgG162 (link) monoclonal antibody was produced in house by secreting hybridoma cell lines and purified from supernatant using Protein A columns (GE Healthcare).
6
Transient Expression of Human Antibodies
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Example 3
Transient gene expression of human antibodies is achieved upon transfection of antibody expression vectors into 293-T human embryonic kidney cells or Chinese Hamster Ovary cells (CHO) using the Polyethylenimine Transfection method (PEI, Polyscience Warrington, USA). After transfection cells are cultured in serum free medium (OPTI-MEM I supplemented with GlutaMAX-I Gibco). Supernatants are collected after 3-6 days of culture and IgG is purified using protein A columns (GE HealthCare, Sweden) on a fast protein liquid chromatography device (FPLC) (GE HealthCare, Sweden).
7
Production and Purification of Monoclonal and Bispecific Antibodies
8
Transient Expression of Human Antibodies
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Example 5
Transient gene expression of human antibodies is achieved upon transfection of antibody expression vectors into 293-T human embryonic kidney cells or Chinese Hamster Ovary cells (CHO) using the Polyethylenimine Transfection method (PEI, Polyscience Warrington, USA). After transfection cells are cultured in serum free medium (OPTI-MEM I supplemented with GlutaMAX-I Gibco). Supernatants are collected after 3-6 days of culture and IgG is purified using protein A columns (GE HealthCare, Sweden) on a fast protein liquid chromatography device (FPLC) (GE HealthCare, Sweden).
9
Engineering Stable and Potent P2X7 Nanobodies
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P2X7-specific nanobodies 13A7, 14D5 and a sequence optimized variant of 1c81 with higher stability and elevated isoelectric point were engineered into bivalent half-life extended nanobodies, or bivalent Fc-fused format, as previously described (Danquah et al., 2016 (link)). Nanobody-constructs were subcloned into the pCSE2.5 expression vector either carrying at the C-terminus a tandem SNB tag (Schaffer et al., 2010 (link)) or carrying at the N-terminus an ALFA-tag (Gotzke et al., 2019 (link)). All nanobody constructs were produced as secretory proteins in transiently transfected HEK-6E cells cultivated in serum-free medium (cells provided by Ives Durocher, Montreal, Canada). After 6 days, cell-supernatants were harvested and clarified by centrifugation. Nanobodies were purified from the supernatants by affinity chromatography using protein A columns (GE Healthcare) and formulated at high concentrations (15–50 mg/ml) in an in-house low aggregation buffer (5.8 mg/ml sodium phosphate monobasic, monohydrate; 1.2 mg/ml sodium phosphate dibasic, anhydrous; 60 mg/ml trehalose; 0.4 mg/ml Tween®; pH 6.5). Integrity and purity were confirmed by SDS-PAGE and Coomassie brilliant blue staining.
10
IgG Purification Using Protein A
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Example 4
Culture supernatants were purified using protein A columns (GE Healthcare/cat#11-0034-95/according to manufacturer's instructions) and eluted in 0.1 M citrate buffer pH 3.0 and immediately neutralized in an equal volume of 1.0 M Tris-HCL pH 8.0 or directly rebuffered to PBS using a desalting column. Alternatively one could purify IgG using protein A beads (sepharose beads CL-4B, GE healthcare cat #170780-01)
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