Antibodies were expressed in FGE-expressing Expi293 cells (a generous gift from Melissa Gray) following Thermo Fischer Scientific’s Expi293 expression protocol. 1 μg (0.5 μg heavy chain and 0.5 μg light chain encoding plasmid) of DNA per mL culture was used for transfection. Antibodies were harvested after 7 days by collecting the supernatant from centrifugation at 300g for 5 min, then centrifugation at 3200g for 30 min at 4 °C. The supernatant was filtered using a 0.22 μm filter unit (Fischer Scientific). Antibodies were purified using Protein A-Sepharose® 4B (Thermo Fisher Scientific) column chromatography in dark. The Protein A beads were packed into Econo-Pac chromatography column (BioRad). The beads were then washed with elution buffer (100 mM Glycine in MQ Water, pH 2.8) and equilibrated under PBS. The filtered supernatant was run through the Protein A column 3 times, washed with PBS 3 times, and eluted with elution buffer into a falcon tube containing 100 μl 1 M Tris buffer, pH 8. Antibody was then buffer exchanged to citrate buffer (50 mM sodium citrate, 50 mM NaCl, pH 5.5) using 30 kDa Amicon Centrifugal Filter.
Protein a sepharose 4b
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protein A Sepharose-4B is a chromatographic resin used for the purification of antibodies and other proteins that bind to Protein A. It consists of Protein A, a bacterial protein, covalently coupled to Sepharose 4B beads. This resin facilitates the capture and separation of target proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Econo pac chromatography column, by Bio-Rad (2 mentions)
0.22 μm filter unit, by Thermo Fisher Scientific (2 mentions)
Laemmli buffer, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 media, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Imject alum adjuvant, by Thermo Fisher Scientific (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Abn161 1, by Merck Group (1 mentions)
Pei max, by Polysciences (1 mentions)
Expifectamine 293 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Pcdna3.4 vector, by Thermo Fisher Scientific (1 mentions)
Hrv 3c protease, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose 4b, by GE Healthcare (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
α phosphoserine peroxidase, by Merck Group (1 mentions)
α gfp, by Roche (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
27 protocols using protein a sepharose 4b
1
Antibody Expression and Purification
2
Antibody Expression and Purification
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Antibodies were expressed in FGE-expressing Expi293 cells (a generous gift from Melissa Gray) following Thermo Fischer Scientific’s Expi293 expression protocol. 1 μg (0.5 μg heavy chain and 0.5 μg light chain encoding plasmid) of DNA per mL culture was used for transfection. Antibodies were harvested after 7 days by collecting the supernatant from centrifugation at 300g for 5 min, then centrifugation at 3200g for 30 min at 4 °C. The supernatant was filtered using a 0.22 μm filter unit (Fischer Scientific). Antibodies were purified using Protein A-Sepharose® 4B (Thermo Fisher Scientific) column chromatography in dark. The Protein A beads were packed into Econo-Pac chromatography column (BioRad). The beads were then washed with elution buffer (100 mM Glycine in MQ Water, pH 2.8) and equilibrated under PBS. The filtered supernatant was run through the Protein A column 3 times, washed with PBS 3 times, and eluted with elution buffer into a falcon tube containing 100 μl 1 M Tris buffer, pH 8. Antibody was then buffer exchanged to citrate buffer (50 mM sodium citrate, 50 mM NaCl, pH 5.5) using 30 kDa Amicon Centrifugal Filter.
3
HEK293 Cell Transfection and Protein Secretion
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HEK293 cells grown in DMEM‐Glutamax (Gibco/Thermo Fisher Scientific) supplemented with 10% FBS (Sigma‐Aldrich) were transfected with calcium phosphate. Media was replaced with fresh media supplemented with 25 μM chloroquine (Sigma‐Aldrich) 1 h before transfection, and again with fresh media without chloroquine 4–6 h after transfection. Forty‐eight hours after transfection, cells were lysed with RIPA buffer supplemented with protease inhibitors. For co‐secretion assays (Figs 1 , 3B and E , and EV1 , EV2 , EV3 ), media was replaced by serum‐free FreeStyle 293 media (Gibco/Thermo Fisher Scientific). The media was harvested 48 h after transfection and centrifuged for 10 min, 1,500 × g, and 4°C to remove cell debris. Protein A Sepharose (4B; Thermo Fisher Scientific) washed three times in PBS buffer was added to each sample (50 µl) and incubated for 2 h at 4°C. Beads were treated with heparinases as described below. For experiments with membrane‐bound proteins (Figs 2C and D and 3G ), HEK293 cells were lysed with RIPA buffer supplemented with protease inhibitors 48 h after transfection. Cell lysates were diluted in IP buffer (100 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM Tris, pH 7.4) and incubated with covalently coupled anti‐HA (Sigma‐Aldrich A2095) or anti‐V5 (Sigma‐Aldrich A7345) beads overnight at 4°C. Beads were treated with heparinases as described below.
4
Immunization of Mice with Schistosoma AChE Subunits
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Three groups of five male BALB/c mice (6-week-old) were intraperitoneally immunized with either SmAChE1, SmBChE1 or SmAChE2 subunits (50 μg/mouse). Antigens were mixed with an equal volume of Imject alum adjuvant (Thermofisher) and administered three times, two weeks apart. Two weeks after the final immunization, mice were sacrificed and blood was collected via cardiac puncture. Blood from all mice in each group was pooled and serum was separated by centrifugation after clotting and stored at −20°C. Polyclonal antibodies were purified from mouse sera using Protein A Sepharose-4B (Thermofisher) according to the manufacturer’s instructions. Serum from naïve mice was similarly processed.
5
Generating wt-ACE2-mFc for Binding Assays
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To generate wt-ACE2-mFc for competitive binding assays, we cloned human ACE2 (1–615) fused to a C-terminal mouse IgG2a Fc into pcDNA3.1. We transfected the plasmid into Expi293 cells for expression. The supernatant was collected and exchanged to 0.1 M sodium phosphate, pH 7.2 and 150 mM NaCl buffer for purification on Protein A Sepharose 4B (ThermoFisher). The protein was eluted in 0.1 M citric acid, pH 3.0 and neutralized in 1 M Tris, pH 9.0 before a final buffer exchange to 25 mM HEPES pH 7.2 and 150 mM NaCl by size-exclusion chromatography with Superose 6 resin (Cytiva). For these studies, we cloned the engineered CDY14HL 1–615 fragment in front of the human IgG1 Fc domain for expression and purification. We previously characterized a decoy fusion to human IgG4 Fc [25 (link)], but found that Fc1 and Fc4 decoy fusions behave similarly with respect to binding and neutralization (e.g., the IC50 values against Wuhan-Hu1 pseudotypes were 37 ng/ml and 35 ng/ml for CDY14HL-Fc4 and CDY14HL-Fc1, respectively).
6
Immunization with Schistosoma Cholinesterases
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Three groups of five male BALB/c mice (6-week-old) were intraperitoneally immunized with either SmAChE1, SmBChE1, or SmAChE2 subunits (50 μg/mouse). Antigens were mixed with an equal volume of Imject alum adjuvant (Thermofisher) and administered three times, two weeks apart. Two weeks after the final immunization, mice were sacrificed and blood was collected via cardiac puncture. Blood from all mice in each group was pooled and serum was separated by centrifugation after clotting and stored at −20 °C. Polyclonal antibodies were purified from mouse sera using Protein A Sepharose-4B (Thermofisher), according to the manufacturer’s instructions. Serum from naïve mice was similarly processed.
7
Immunoprecipitation of Neurexin and CA10
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Cell lysates or brain homogenates were diluted (equal amount of total protein in the different samples) in IP buffer (100 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM Tris, pH 7.4). For immunoprecipitations from cell lysates of primary cortical cultures (Fig 2F ), 5 µg of primary antibody: pan‐neurexin (rabbit, ABN161‐I; Merck) was used. Protein A Sepharose (4B; Thermo Fisher Scientific) washed three times in IP buffer was added to each sample (50 µl) and incubated for 2 h at 4°C. For immunoprecipitations from brain lysates (Figs 6C and 2I ) 5 µg of primary antibody: pan‐neurexin (rabbit, ABN161‐I; Merck) or polyclonal antisera directed against recombinant CA10 (rabbit “Hadlai”, Agrisera) were conjugated to 50 µl of Protein A/G magnetic beads (Pierce/Thermo Fisher Scientific) using bis[sulfosuccinimidyl] suberate (BS3) (Thermo Fisher Scientific) according to manufacturer’s protocol. Briefly, beads were incubated with the antibodies overnight at 4°C, washed with 0.15 M NaCl and incubated with 5 mM BS3 for 30 min at room temperature. The reaction was quenched by adding 50 mM Tris and incubating 15 min at room temperature. Beads were washed with IP buffer and incubated with the samples overnight. Heparinase treatment followed.
8
Production and Purification of LDLRAD3-D1-Fc
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LDLRAD3-D1-Fc was produced using Expi293F cells, as described (Ma et al., 2020 (link)). Briefly, codon-optimized vectors encoding the LDLRAD3-D1-Fc (human IgG1 or murine IgG2b) proteins were cotransfected with receptor-associated protein, an LDL-receptor family protein chaperone. Protein was purified from supernatant using protein A Sepharose 4B (Thermo Fisher Scientific) and dialyzed into D-PBS with 1 mM CaCl2 and EDTA-free protease inhibitors (Roche). Purity was confirmed by SDS-PAGE, and protein was stored at 4°C for subsequent use.
9
Immunoprecipitation of Transfected Proteins
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The transfected HEK293T cells were harvested at 48 h posttransfection for the immunoprecipitation (IP) experiments. The cells were washed once with cold PBS, lysed in NP-40 lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 0.5% NP-40, protease inhibitor cocktail from Roche), and passed through a 23-gauge needle 10 to 15 times, and then the cell lysates were incubated on ice for 15 min. After centrifugation, the cell lysates were subjected to preclearing using protein A-Sepharose 4B (ThermoFisher) for 2 h at 4°C and then incubated with antibodies overnight. The next day, protein A/G XPure agarose resin was added to the lysates, which were further incubated for 2 h at 4°C. The IPs were washed three times with the lysis buffer and then resuspended in 2× Laemmli buffer (Bio-Rad). The IP samples along with the input samples were analyzed by immunoblot.
10
Immunoprecipitation of Transfected Proteins
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The transfected HEK293T cells were harvested at 48 h posttransfection for the immunoprecipitation (IP) experiments. The cells were washed once with cold PBS, lysed in NP-40 lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 0.5% NP-40, protease inhibitor cocktail from Roche), and passed through a 23-gauge needle 10 to 15 times, and then the cell lysates were incubated on ice for 15 min. After centrifugation, the cell lysates were subjected to preclearing using protein A-Sepharose 4B (ThermoFisher) for 2 h at 4°C and then incubated with antibodies overnight. The next day, protein A/G XPure agarose resin was added to the lysates, which were further incubated for 2 h at 4°C. The IPs were washed three times with the lysis buffer and then resuspended in 2× Laemmli buffer (Bio-Rad). The IP samples along with the input samples were analyzed by immunoblot.
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