After fusion with HMMA2.5 myeloma cells, hybridomas producing MARV-specific antibodies were cloned biologically by two rounds of limiting dilution and by single-cell fluorescence-activated cell sorting. After cloning, hybridomas were expanded in post-fusion medium (ClonaCell-HY Medium E, STEMCELL Technologies #03805) until 50% confluent in 75-cm2 flasks (Corning #430641). For antibody production, cells from one 75-cm2 flask were collected with a cell scraper and expanded to four 225-cm2 flasks (Corning #431082) in serum-free medium (Hybridoma-SFM, Gibco #12045-076). After 21 days, supernates were clarified by centrifugation and sterile filtered using 0.2-μm pore size filter devices. HiTrap Protein G or HiTrap MabSelectSure columns (GE Healthcare Life Sciences #17040501 and #11003494 respectively) were used to purify antibodies from filtered supernates. Fab fragments were generated by papain digestion (Pierce Fab Preparation Kit, Thermo Scientific #44985) and purified by chromatography using a two-column system where the first column contained protein G resin (GE Healthcare Life Sciences #29048581) and the second column contained either anti-kappa or anti-lambda antibody light chain resins (GE Healthcare Life Sciences #17545811 and #17548211 respectively).
Protein g resin
Manufactured by GE Healthcare
Sourced in United States
Protein G resin is a chromatography material used for the purification of antibodies. It consists of Protein G, a bacterial protein that binds to the Fc region of immunoglobulins, immobilized on a solid support matrix. The resin can be used to capture and purify a variety of antibody types from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap protein g, by GE Healthcare (1 mentions)
Clonacell hy medium e, by STEMCELL (1 mentions)
Hybridoma sfm, by Thermo Fisher Scientific (1 mentions)
Pierce fab preparation kit, by Thermo Fisher Scientific (1 mentions)
Hitrap mabselectsure columns, by GE Healthcare (1 mentions)
Balb c mice, by Nara Biotech (1 mentions)
Superose 6 increase 10 300 column, by GE Healthcare (1 mentions)
Papain agarose resin, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Un scan it software, by Silk Scientific (1 mentions)
Polymyxin b sulfate, by Merck Group (1 mentions)
Polymyxin b, by Merck Group (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
96 well depletion plates, by GE Healthcare (1 mentions)
Mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Etoposide, by Merck Group (1 mentions)
16 protocols using protein g resin
1
Monoclonal Antibody Production and Purification
2
Establishment of Integrated Capture Assay
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For establishment of ICA kit, the monoclonal antibody, anti-PED 3B12-1A6, purified from ascetic fluid using protein G resin (GE Healthcare) was coated onto a specific area (test line) of a nitrocellulose membrane, while goat antimouse IgG was coated onto another specific area (control line) on the same membrane. To produce the test conjugate, a different monoclonal antibody, anti-PED 1H12-1C6, was mixed with colloidal gold prepared by trisodium citrate reduction method as previously described,12 (link) and then the antibody mixture was treated by a previously described method.12 (link) The assay strips were prepared by assembly with the colloidal gold-conjugated glass fibre, the nitrocellulose membrane and an absorbent paper using polyvinyl chloride self-adhesive floor.
3
Generation of Monoclonal Antibodies against mRID-cHA
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Monoclonal antibodies against the mRID-cHA stalks were generated by murine cell fusion/hybridoma19 by ATGen (Seongnam, Republic of Korea). All animal research was performed according to the guidelines of Ministry of Food and Drug Safety of Republic of Korea. All the experiments were approved by ATGen Institutional Animal Care and Use Committee (IACUC; permit number: ATGen2016-0113-06). BALB/c mice were purchased from NARA Biotech (Seoul, Republic of Korea). 8-weak-old female mice were immunized two times at two-weeks interval. Screening for hybridoma clones from the sacrificed mice that were positive to the mRID-cHA stalk protein was done using ELISA. Selected positive clones were purified using Protein G resin (GE Healthcare, Chicago, IL) and dialyzed against PBSA (PBS with 0.05% sodium azide).
4
Isolation and Fab Preparation from IgG
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For IgG isolation, 1 ml of human serum diluted to 5 ml with PBS was incubated with 500 μl of washed protein G resin (GE Healthcare) overnight at 4°C. The resin was washed three times with PBS and eluted with 10 ml of 0.1 M glycine buffer at pH 2.5. The eluate was immediately neutralized with 4 ml of 1 M tris-HCl (pH 8.0) and buffer-exchanged to PBS using 100-kDa cutoff Amicon ultrafiltration units. For Fab preparation, 4 mg of concentrated polyclonal IgG samples was incubated with papain-agarose resin (Thermo Fisher Scientific) in digestion buffer (20 mM sodium phosphate, 10 mM EDTA, and 20 mM cysteine, pH 7.4) for around 22 hours in a 37°C incubator. The digest was removed from the beads and buffer-exchanged to PBS. The undigested IgGs were removed by SEC using a Superose 6 increase 10/300 column (GE Healthcare Biosciences). The purified Fabs were concentrated and assessed by SDS–polyacrylamide gel electrophoresis for purity.
5
Immunoprecipitation and Immunoblotting Protocol
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Tissues were lysed in lysis buffer A [50 mM Hepes-NaOH (pH 7.5), 3 mM MgCl2, 150 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethane sulfonylfluoride, leupeptin (1 μg/ml), 1 mM EDTA, 1 mM Na3VO4, and 10 mM NaF] containing biochemical detergents (0.5% NP-40, 1% CHAPS, and 0.03% SDS). Unless otherwise indicated, all lysis steps were performed at 4°C (12 (link), 36 (link)). For immunoprecipitations, the supernatants cleared by centrifugation were mixed with an antibody-absorbed protein G resin (GE Healthcare) or ExtraCruz (Santa Cruz Biotechnology) according to the respective manufacturer’s instructions. The immunoprecipitates or proteins in the cell supernatants were denatured, subjected to SDS–polyacrylamide gel electrophoresis, and blotted to polyvinylidene difluoride membranes using the TransBlot TurboTransfer System (Bio-Rad). The membranes were blocked with Blocking One (Nacalai) and immunoblotted using a primary antibody followed by a peroxidase-conjugated secondary antibody. The bound antibodies were detected using Chemiluminescence One (standard and strong detection reagents; Nacalai). The bands were scanned using a C-DiGit Blot Scanner (MS Systems). They were densitometrically analyzed to identify their quantification using UN-SCAN-IT software (Silk Scientific).
6
Recombinant Fab Expression and Purification
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After screening, the DNA fragment of the selected clones was inserted into the modified pComb3XSS vector with a C-terminus 6× His-tag and Flag-tag. To express the Fab, the vector was transformed into E. coli HB2151 cells, which were grown at 37 °C in SB medium to an optical density of A600 ~0.6–0.8. Expression was induced with isopropyl-1-thio-β-d -glucopyranoside (IPTG) (1 mM) at 30 °C for 12–16 h. To prepare the periplasmic extract, the bacterial cells were pelleted and resuspended in PBS. polymyxin B sulfate (Sigma‒Aldrich, 0.5 mu/ml) was added to the suspension at a ratio of 1:1000 (volume of polymyxin B: culture volume). After 1 h of incubation at room temperature, followed by centrifugation, the cell lysate was collected, and the Fab was purified using Ni-NTA spin (QIAGEN). For protein production measurement by cryo-electron microscope analysis, after the Ni-NTA spin purification, the Fab protein was further purified by protein G resin (GE Healthcare).
7
Immunoprecipitation of Cellular Proteins
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Supernatants of the cell lysates in buffer A were used for immunoprecipitation of the purposed proteins [15 (link),16 (link)]. The supernatants were mixed with protein G resin (GE Healthcare, Fairfield Easton, CT, USA) that had been absorbed with an antibody. The immunoprecipitates in supernatants of the cell lysate were denatured, subjected to polyacrylamide gel electrophoresis, and blotted onto polyvinylidene difluoride membranes for immunoblotting.
For immunoprecipitation of the lysosome, we used buffer B (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF) and homogenized cell lysates with Potter-Elvehjem homogenizer. The homogenates were centrifuged at 150× g for 10 min using a tabletop centrifuge. The supernatants were gently mixed with an anti-SLC38A9 antibody-absorbed protein G resin [17 (link)]. The immunoprecipitates were denatured, subjected to polyacrylamide gel electrophoresis, and blotted onto membranes for immunoblotting.
For immunoprecipitation of the lysosome, we used buffer B (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF) and homogenized cell lysates with Potter-Elvehjem homogenizer. The homogenates were centrifuged at 150× g for 10 min using a tabletop centrifuge. The supernatants were gently mixed with an anti-SLC38A9 antibody-absorbed protein G resin [17 (link)]. The immunoprecipitates were denatured, subjected to polyacrylamide gel electrophoresis, and blotted onto membranes for immunoblotting.
8
Antibody Production from Sorted B Cells
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The 96-well plates containing sorted B cells were thawed on the ice and used for RT-PCR (Qiagen OneStep RT-PCR Kit) followed by nested PCR with primers described in a previous study [38 (link)]. After analysis of the VDJ sequence by IMGT, the VH and VK fragments were amplified by PCR and subjected to appropriate restriction enzyme digestion. The VH genes were cloned into a modified expression vector with signal peptide and the human constant region of IgG1. The VK genes were cloned into a modified expression vector with a signal peptide and the human kappa chain constant region. The VH and VK plasmids were co-transfected into Expi293F cells (Thermo Scientific) for antibody production. After 5 days of culture, the individual antibodies in the culture supernatant were purified using protein G resin (GE healthcare). The antibodies were replaced into PBS and analyzed by SDS-PAGE.
9
Antibody Purification from Hybridoma Supernatant
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Culture supernatant from hybridoma were concentrated by adding 50/45 (v/v) of saturated ammonium sulfate (DAEJUNG) and kept at 4°c for overnight. Precipitates were reconstituted with PBS and then dialyzed for three times against 0.1 M sodium acetate (pH 5.0). Antibodies were purified using protein G resin (GE Healthcare) according to the protocol from the manufacturer.
10
Immunoprecipitation of Lysosome Proteins
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Supernatants of the cell lysates in buffer A were used for immunoprecipitation of the purposed proteins as described elsewhere [16 (link),17 (link)]. The supernatants were mixed with protein G resin (GE Healthcare, Fairfield Easton, CT, USA) that had been absorbed with an antibody. The immunoprecipitates in supernatants of the cell lysate were denatured, subjected to denaturing polyacrylamide gel electrophoresis, and blotted onto polyvinylidene difluoride membranes for immunoblotting.
For immunoprecipitation of the lysosome [18 (link)], we used buffer B (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF) and homogenized cell lysates with Potter-Elvehjem homogenizer. The homogenates were centrifuged at 150× g for 10 min in a tabletop centrifuge. The supernatants were gently mixed with an anti-SLC38A9 antibody-absorbed protein G resin according to their respective manufacturer’s instructions. The immunoprecipitates were denatured, subjected to denaturing gel electrophoresis, and blotted for immunoblotting.
For immunoprecipitation of the lysosome [18 (link)], we used buffer B (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF) and homogenized cell lysates with Potter-Elvehjem homogenizer. The homogenates were centrifuged at 150× g for 10 min in a tabletop centrifuge. The supernatants were gently mixed with an anti-SLC38A9 antibody-absorbed protein G resin according to their respective manufacturer’s instructions. The immunoprecipitates were denatured, subjected to denaturing gel electrophoresis, and blotted for immunoblotting.
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