The gene encoding the antibody scFv domain was cloned into a pFUSE-Fc expression vector (Cat. No. pfuse-hg1fc2, Invivogen, Hong Kong) to generate abEC1.1 as a diabody-Fc fusion protein (Wu et al., 2001 (link)) which comprises the entire Fc domain of human immunoglobulin G1 (IgG1) (Silverton et al., 1977 (link); Bujak et al., 2014 (link); Frenzel et al., 2016 (link)). In addition, the pFUSE-Fc expression vector was modified to generate a variant of the antibody with a murine Fc (abEC1.1m). For antibody production, a FreeStyleTM 293-F cell line (Thermo Fisher Scientific, Cat. No. R79007), maintained in Freestyle 293 Expression Medium (Thermo Fisher Scientific, Cat. No. 12338026), was stably transfected with the abEC1.1 or abEC1.1m expression vector. Expressed antibodies were purified using HiTrap Protein A HP columns (GE Healthcare, Cat. No. 17-0403-03) with the ÄKTApurifier 100 system (GE Healthcare). After purification, the buffer was exchanged to PBS (pH 7.4) and the antibodies were kept in PBS at 4°C.
Pfuse hg1fc2
Manufactured by InvivoGen
Sourced in Hong Kong
The Pfuse-hg1fc2 is a laboratory equipment product designed for cell fusion. It facilitates the fusion of cells, a crucial process in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (4 mentions)
Hitrap protein a hp column, by GE Healthcare (2 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (2 mentions)
Ktapurifier 100 system, by GE Healthcare (2 mentions)
Pfuse higg1 fc2, by InvivoGen (2 mentions)
Freestyle 293 f cell line, by Thermo Fisher Scientific (1 mentions)
Ktaxpress purifier, by GE Healthcare (1 mentions)
Hitrap protein g column, by GE Healthcare (1 mentions)
5 protocols using pfuse hg1fc2
1
Antibody scFv Diabody-Fc Fusion Protein Production
2
Expression and Purification of scFv-Fc and Full-Length Antibodies
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DNA sequences encoding the candidate scFv antibodies were cloned into a pFuse expression vector (#pfuse-hg1fc2; InvivoGen) for expression of scFv-Fc proteins with the entire Fc domain of human IgG1. For antibodies in the full-length IgG1 format, variable regions of heavy chain and light chain (VH and VL) from the scFv sequence were cloned into plasmids with the complete constant domains of IgG1 heavy chain and light chains (CH and CL). The antibodies were expressed through transfection of the scFv-Fc expression plasmid, or co-transfection of equal molar of heavy chain and light plasmids for full-length antibody, into HEK293F cells followed by cell culture for 5 days. Antibodies in the medium were purified with a HiTrap Protein A HP column (#17-0403-03; GE Healthcare) by ÄKTAxpress purifier (GE Healthcare). Purified antibodies were concentrated and stored in PBS buffer (pH 7.4) at −80 °C.
3
VH Domain Antibody Fusion Protein
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Rabbit VH domain antibodies were fused with His-FLAG tag, inserted into the expression vector pFUSE-hIgG1-Fc2 (pfuse-hg1fc2; InvivoGen), and expressed as a VH-His-FLAG-hFc format in 293F cells according to the aforementioned method. The 6× His tag was used for Nickel column affinity purification and the hFc tag was used in the cell binding assay by flow cytometry that used goat anti-human polyclonal antibody (cat. no. 109-135-170, Jackson ImmunoResearch Laboratories, Inc.) as secondary antibody.
4
Cloning and Production of scFv-Fc Fusion Proteins
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The gene encoding the Cx-binding scFv complex [35] (link) was inserted into a cloning plasmid (Cat. No. pfuse-hg1fc2, Invivogen, Hong Kong) to generate a scFv-Fc fusion protein comprising constant hinge, CH2 and CH3 domains of human secreted IgG1 [46] (link). A similar cloning plasmid (Cat. No. pfuse-mg1fc2, Invivogen) was used to generate a chimeric version with constant hinge, CH2 and CH3 domains of mouse secreted IgG1 (see e.g. UniProtKB – P01868). For antibody production, a FreeStyle™ 293-F cell line (Cat. No. R79007, ThermoFisher Scientific, Waltham, MA, U.S.A.), maintained in Freestyle 293 Expression Medium (Cat. No. 12338026, ThermoFisher Scientific), was stably transfected with the expression vector of either scFv-Fc polypeptide. Cells were tested periodically with the MycoFluor™ Mycoplasma Detection Kit (Cat. No. M7006, ThermoFisher Scientific) to exclude contamination. Expressed antibodies were purified using HiTrap MabSelectTM columns (Cat. No. 28-4082-53, GE Healthcare, Pittsburgh, PA, USA) with the ÄKTApurifier 100 system (GE Healthcare) following the Manufacture's instruction. After purification, the buffer was exchanged to phosphate buffer saline (PBS, pH 7.4) and the antibodies were kept in PBS at 4°C [36] (link).
5
Generation of CLR-hFc Fusion Proteins
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CLR-hFc fusion proteins were generated as previously described [31 (link),64 (link)]. Briefly, extracellular domain sequences of the respective CLRs were inserted into an expression vector containing the Fc part of human IgG1 (pFUSE-hIgG1-Fc2, Invivogen, CAT#pfuse-hg1fc2, San Diego, CA, USA) and CHO-S cells were transiently transfected with the CLR-hFc-expression vector using polyethylenimine (PEI, Polysciences, CAT#23966-2, Hirschberg an der Bergstrasse, Germany). CLR-hFc fusion proteins were purified from the supernatant by affinity chromatography via HiTrap protein G columns (GE Healthcare, CAT#17-0404-01, Chicago, IL, USA). SDS-PAGE with subsequent hFc-detecting immunoblot were applied to confirm the purity and identity of obtained fusion proteins. Modular design of these fusion proteins enabled the use of CLR-hFc chimeras for detection of CLR/Toxocara spp. interactions in ELISA-, fluorescence microscopy- and immunoblot-based methods.
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