The creation of a mammalian expression plasmid for EGFP‐tagged ZO‐1 (ZO‐1‐GFP) has been described previously.14 Expression plasmids for NH2‐terminal HA‐tagged claudin‐3 and claudin‐3ΔYVF were constructed by inserting each cDNA of claudin‐3 and claudin‐3ΔYVF into a pHA‐C3 vector, which was constructed by replacing the EGFP cDNA with the HA epitope sequence in the pEGFP‐C3 vector (Clontech). HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and were cotransfected with ZO‐1‐GFP and HA‐claudin‐3 or HA‐claudin‐3ΔYVF using Avalanche Everyday Transfection Reagents (EZ Biosystems). HEK293 cells were then cultured for 2 days and lysed with ice‐cold lysis buffer (50 mM Tris‐HCl, pH 7.4, 150 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM DTT, and a protease inhibitor cocktail [#25955; Nacalai Tesque]) and incubated on ice for 20 min. Cell lysates were clarified by centrifugation at 18,000 × g for 15 min and preincubated with Protein‐G‐Sepharose™ 4 Fast Flow (#17061801; Cytiva) for 1 h. Supernatants were incubated for 2 h at 4°C with an anti‐GFP antibody and Protein‐G‐Sepharose™ 4 Fast Flow. Immunoprecipitates were subjected to SDS‐PAGE and analyzed by immunoblotting with an anti‐HA antibody.
Protein g sepharose 4 fast flow
Manufactured by Cytiva
Sourced in United States, United Kingdom
Protein G Sepharose 4 Fast Flow is a chromatography medium used for the purification of antibodies. It consists of recombinant Protein G covalently coupled to Sepharose 4 Fast Flow, a highly cross-linked agarose matrix. This medium is designed for efficient and rapid purification of immunoglobulins from various sources.
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Lab products found in correlation
Amicon ultra 4 centrifugal filter, by Merck Group (5 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (4 mentions)
Millex gp filter, by Merck Group (3 mentions)
Pei max, by Polysciences (3 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Expi293f expression system, by Thermo Fisher Scientific (1 mentions)
Pd minitrap g 25, by Cytiva (1 mentions)
Pegfp c3 vector, by Takara Bio (1 mentions)
Normal rabbit igg, by Merck Group (1 mentions)
Branched polyethylenimine, by Merck Group (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
0.22 m filter, by Merck Group (1 mentions)
Hek293 cells, by Merck Group (1 mentions)
Expi293f, by Thermo Fisher Scientific (1 mentions)
Thp 1 monocytic cells, by Merck Group (1 mentions)
Peg1500, by Roche (1 mentions)
27 protocols using protein g sepharose 4 fast flow
1
Co-Immunoprecipitation of ZO-1 and Claudin-3
2
VNAR Antibody Expression and Purification
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VNAR cDNAs prepared by gene synthesis were fused with human Fc or AGIA-His tandem tag and subcloned into the pcDNA3.4 expression vector using Gibson Assembly. VNAR antibodies were expressed using the Expi293F Expression System (Thermo Fisher Scientific, A14635), according to the manufacturer’s instructions. VNAR-Fc antibodies were purified using protein G Sepharose 4 Fast Flow (Cytiva), and then the buffer was exchanged with PBS using PD MiniTrap G-25 (Cytiva). VNAR-AGIA-His antibodies were purified via Ni Sepharose High Performance, and then buffer was exchanged with PBS via PD-10 column. Antibody concentration was determined using the extinction coefficient method (Gasteiger et al., 2005 (link)) with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Purified antibodies were frozen and stored at −30°C.
3
Cloning and Expression of Monoclonal Antibodies
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Antibody genes were sequenced, cloned and expressed as previously reported50 (link)–52 (link). Briefly, reverse transcription of RNA from FACS-sorted single cells was performed to obtain complementary DNA, which was then used for amplification of the immunoglobulin IGH, IGK and IGL genes by nested PCR. Amplicons from this first PCR reaction served as templates for sequence- and ligation-independent cloning into human IgG1 antibody expression vectors. Monoclonal antibodies were produced by transiently transfecting Expi293F cells cultured in Freestyle-293 Expression Medium (ThermoFisher) with equal amounts of immunoglobulin heavy and light chain expression vectors using polyethyleneimine Max (PEI-MAX, Polysciences) as a transfection reagent. After 6–7 d of culture, cell supernatants were filtered through 0.22-μm Millex-GP filters (Merck Millipore), and antibodies were purified using Protein G Sepharose 4 Fast Flow (Cytiva) according to the manufacturer’s instructions and buffer-exchanged and concentrated in PBS by Amicon Ultra-4 centrifugal filters (30-kDa cutoff, Millipore). Where indicated, the monoclonal antibody against Zika virus Z02150 was used as an isotype control.
4
Mapping Arc protein interactions
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HEK293FT cells were co-transfected with plasmids expressing mSc-ArcNb H11 (mSc-H11) and an mTurquoise2 (mTq2)-tagged Arc construct. mTq2 was N-terminally fused to 1) Arc full-length (FL), 2) N-terminal region (NTR 1–140), 3) linker (135–216), 4) C-terminal region (CTR 208–396), 5) N-lobe (NL 208–277), or 6) C-lobe + C-terminal tail (CL + tail 278–396). Transfected cells were lysed in M-RIPA buffer. First, 2 μg of normal rabbit IgG (Merck, Cat# 12–370, RRID:AB_145841), anti-mCherry (mCh) antibody against mSc-H11, or anti-Arc rabbit polyclonal antibody were absorbed to Protein G Sepharose 4 Fast Flow (Cytiva) for 1 h at room temperature. Then, antibody/bead complexes were mixed with the cell lysates overnight at 4 °C for co-immunoprecipitation (co-IP). The sepharose beads were washed four times with M-RIPA buffer and resuspended in 2X Laemmli sample buffer containing 100 mM DTT before denaturing at 95 ºC for 5 min. The supernatants were subjected to SDS-PAGE followed by immunoblot analysis.
5
Recombinant Antibody Production in 293-6E Cells
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All antibodies were produced as previously described (Mouquet et al., 2011 (link)). Briefly, 293-6E cells that were maintained in Freestyle 293 Expression Medium (Thermo Fisher Scientific) were transiently transfected by using equal amounts of Ig heavy and light chain expression vectors using branched polyethylenimine (Sigma). 7 d after transfection, cells were spun down at 4,200 g for 40 min at 4°C, and supernatants were filtered through a 0.22-µM filter (Millipore). Antibodies were purified from filtered supernatants using Protein G Sepharose 4 Fast Flow (Cytiva). Antibodies were buffer exchanged and concentrated into PBS using an Amicon Ultra centrifugal filter (Millipore).
6
Monoclonal Antibody Production from Single Cells
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Antibody genes were sequenced, cloned and expressed as previously reported 79 (link), 80 (link), 81 (link). Briefly, reverse-transcription of RNA from FACS-sorted single cells was performed to obtain cDNA, which was then used for amplification of the immunoglobulin IGH, IGK and IGL genes by nested PCR. Amplicons from this first PCR reaction served as templates for sequence and ligation independent cloning (SLIC) into human IgG1 antibody expression vectors. Monoclonal antibodies were produced by transiently transfecting Expi293F cells cultured in Freestyle-293 Expression Medium (ThermoFisher) with equal amounts of Ig heavy and light chain expression vectors using polyethylenimine Max (PEI-MAX, Polysciences) as a transfection reagent. After 6–7 days of culture, cell supernatants were filtered through 0.22 μm Millex-GP filters (Merck Millipore), and antibodies were purified using Protein G Sepharose 4 Fast Flow (Cytiva) according to manufacturer’s instructions and buffer-exchanged and concentrated in PBS by Amicon Ultra-4 centrifugal filters (30 kDa cutoff, Millipore). Where indicated, the anti-Zika virus monoclonal antibody Z021 79 (link) was used as an isotype control.
7
Porcine Polyclonal Antibody Production
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Porcine polyclonal antibodies were prepared by Zhengzhou University using the SARS-CoV-2 protein subunit vaccine (Chinese hamster ovary (CHO) cell). Porcine polyclonal antibody was purified by affinity chromatography using Protein G Sepharose 4 Fast Flow (Cytiva, 17061806, Wilmington, DE, USA).
8
Antibody Production in Cell Lines
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THP-1 monocytic cells (Sigma Aldrich, 88081201-1VL) were cultured in RPMI with 10% FBS and L-glutamine and were kept at 2.5-10 × 105 cells per mL. HEK293 cells (Sigma Aldrich, 12022001‐1VL) and Expi293F (Thermofisher, A14527) cells were used to produce antibodies similarly as done before37 (link),38 (link). Supernatant containing produced antibodies was purified through incubation with protein G sepharose 4 fast flow (Cytiva, 17-0618-05) according to the manufacturer’s instruction.
9
Generation of Monoclonal Antibodies Against rE2 Antigen
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BALB/c mice were injected intraperitoneally with rE2 (100 μg/dose); Imject Alum (Thermo Fisher Scientific) was used as an adjuvant. After three booster immunizations at 1-week intervals,
splenocytes were harvested and then fused with P3U1 cells by using PEG1500 (Roche Diagnostics, Indianapolis, IN, USA). The resulting hybridomas were grown in RPMI 1640 medium supplemented
with 10% FBS and hypoxanthine–aminopterin–thymidine selection medium supplement (Thermo Fisher Scientific). Hybridomas were chosen on the basis of the reactivity of their culture
supernatants against rE2 in ELISAs. We established two hybridoma clones: E2-1a-1 and E2-1a-6. The reactivity of these mAbs to the antigen was confirmed by Western blot analysis and
immunofluorescence staining. Protein G Sepharose 4 Fast Flow (Cytiva, Marlborough, MA, USA) was used to purify the mAbs from the supernatants of hybridomas cultured in Hybridoma-SFM medium
(Thermo Fisher Scientific).
splenocytes were harvested and then fused with P3U1 cells by using PEG1500 (Roche Diagnostics, Indianapolis, IN, USA). The resulting hybridomas were grown in RPMI 1640 medium supplemented
with 10% FBS and hypoxanthine–aminopterin–thymidine selection medium supplement (Thermo Fisher Scientific). Hybridomas were chosen on the basis of the reactivity of their culture
supernatants against rE2 in ELISAs. We established two hybridoma clones: E2-1a-1 and E2-1a-6. The reactivity of these mAbs to the antigen was confirmed by Western blot analysis and
immunofluorescence staining. Protein G Sepharose 4 Fast Flow (Cytiva, Marlborough, MA, USA) was used to purify the mAbs from the supernatants of hybridomas cultured in Hybridoma-SFM medium
(Thermo Fisher Scientific).
10
Purification and Chemotaxis Assessment of COVID-19 IgGs
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IgGs were purified from a subset of samples of the COVID-19 and healthy control cohorts using Protein G Sepharose 4 Fast Flow (Cytiva) according to the manufacturer’s instructions (plasma/resuspended beads at a 5:4 (v/v) ratio), buffer-exchanged and concentrated in PBS by Amicon Ultra-4 centrifugal filters (30-kDa cutoff, Millipore). Chemotaxis of preB 300.19 expressing CCR2 or CXCR1 was performed at a final IgG concentration of 200 µg ml−1 (IgG concentration in human serum: ~10,000 µg ml−1 (ref. 29 (link))), in the presence of the chemokine concentration resulting in peak migration when no antibodies were added (CCL7 (100 nM), CCL8 (100 nM), CXCL8 (1 nM)) (Extended Data Fig. 8k ).
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