Pfuse2ss clig hl2

Manufactured by InvivoGen

PFUSE2ss-CLIg-hl2 is a lab equipment product. It is a fusion protein that combines the soluble portion of the human Fc receptor with the human IgG1 hinge, CH2, and CH3 domains. This fusion protein can be used for various research applications.

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4 protocols using pfuse2ss clig hl2

Generation and Characterization of Regdanvimab Antibodies

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The Regdanvimab and its analogs DNA sequences variable regions were synthesized in GenScript. The heavy and light chain variable regions were inserted into pfuse2ss-chig-hg1 and pfuse2ss-clig-hl2 (InvivoGen) vector accordingly. Antibodies were produced using ExpiCHO cells (Thermo Fisher). 8 days after transfection, antibodies were purified from medium using Protein A/G resin (Thermo Fisher). After dialysis in PBS, antibodies were used in neutralization assay. Antibodies’ concentrations were measured by BCA (Bicinchoninic Acid) Protein Assay (Thermo Fisher).

Beshnova D., Fang Y., Du M., Sun Y., Du F., Ye J., Chen Z.J, & Li B. (2022). Computational approach for binding prediction of SARS-CoV-2 with neutralizing antibodies. Computational and Structural Biotechnology Journal, 20, 2212-2222.

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Antibody Expression and Purification

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For each antibody, variable genes were inserted into plasmids encoding the constant region for the heavy chain (pFUSEss-CHIg-hG1, Invivogen) and light chain (pFUSE2ss-CLIg-hl2, Invivogen and pFUSE2ss-CLIg-hk Invivogen) and synthesized from GenScript. mAbs were expressed in FreeStyle 293F or Expi293F mammalian cells (ThermoFisher) by co-transfecting heavy chain and light chain expressing plasmids using polyethylenimine (PEI) transfection reagent and cultured for 5–7 days. FreeStyle 293F (ThermoFisher) and Expi293F (ThermoFisher) cells were maintained in FreeStyle 293F medium or FreeStyle F17 expression medium supplemented with 1% of 10% Pluronic F-68 and 20% of 200 mM L-Glutamine. These cells were cultured at 37°C with 8% CO ₂ saturation and shaking. After transfection and 5–7 days of culture, cell cultures were centrifuged at 6000 rpm for 20 minutes. Supernatant was 0.45 μm filtered with Nalgene Rapid Flow Disposable Filter Units with PES membrane. Filtered supernatant was run over a column containing Protein A agarose resin that had been equilibrated with PBS. The column was washed with PBS, and then antibodies were eluted with 100 mM Glycine HCI at pH 2.7 directly into a 1:10 volume of 1 M Tris-HCI pH 8. Eluted antibodies were buffer exchanged into PBS 3 times using 10kDa Amicon Ultra centrifugal filter units.

Setliff I., Shiakolas A.R., Pilewski K.A., Murji A.A., Mapengo R.E., Janowska K., Richardson S., Oosthuysen C., Raju N., Ronsard L., Kanekiyo M., Qin J.S., Kramer K.J., Greenplate A.R., McDonnell W.J., Graham B.S., Connors M., Lingwood D., Acharya P., Morris L, & Georgiev I.S. (2019). High-throughput mapping of B-cell receptor sequences to antigen specificity. Cell, 179(7), 1636-1646.e15.

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Mammalian Expression and Purification of Monoclonal Antibodies

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For each antibody, variable genes were inserted into plasmids encoding the constant region for the heavy chain (pFUSEss-CHIg-hG1, Invivogen) and light chain (pFUSE2ss-CLIg-hl2, Invivogen and pFUSE2ss-CLIg-hk, Invivogen) and synthesized from GenScript. mAbs were expressed in FreeStyle 293F or Expi293F mammalian cells (ThermoFisher) by co-transfecting heavy chain and light chain expressing plasmids using polyethylenimine (PEI) transfection reagent and cultured for 5-7 days. FreeStyle 293F (ThermoFisher) and Expi293F (ThermoFisher) cells were maintained in FreeStyle 293F medium or FreeStyle F17 expression medium supplemented with 1% of 10% pluronic F-68 and 20% of 200 mM L-Glutamine. These cells were cultured at 37°C with 8% CO₂ saturation and shaking. After transfection and 5-7 days of culture, cell cultures were centrifuged at 6000 rpm for 20 minutes. Supernatant was 0.45 μm filtered with PES membrane Nalgene Rapid Flow Disposable Filter Units. Filtered supernatant was run over a column containing Protein A agarose resin that had been equilibrated with PBS. The column was washed with PBS, and then antibodies were eluted with 100 mM Glycine HCl at pH 2.7 directly into a 1:10 volume of 1 M Tris-HCl pH 8. Eluted antibodies were buffer exchanged into PBS three times using 10 kDa or 30 kDa Amicon Ultra centrifugal filter units.

Murji A.A., Raju N., Qin J.S., Kaldine H., Janowska K., Fechter E.F., Mapengo R., Scheepers C., Setliff I., Acharya P., Morris L, & Georgiev I.S. (2021). Sequence and functional characterization of a public HIV-specific antibody clonotype. iScience, 25(1), 103564.

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Single-cell antibody variable gene amplification

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cDNA was synthesized from mRNA of single cells as follows: Sorted cells were incubated with 200ng Random Hexamer Primer, 4 μL 5x RT Buffer (Thermo Fisher Scientific), 1 μL dNTPs mix (10 mM, Promega), 1 μL DTT (Thermo Fisher Scientific), 8U RNasin Ribonuclease Inhibitor (Promega), 0.25 μL Superscript III (200 U/mL, Thermo Fisher Scientific), and 6.5 μL of RNase-free H2O at 42°C for 10 min, 25°C for 10 min, 50°C for 60 min, and 94°C for 5 min⁶⁷ (link) and antibody variable genes were amplified as described previously¹² (link) with minor modifications. Briefly, antibody heavy gamma (γ) and light lambda (λ) variable genes were amplified independently in nested PCR using MyTaq HS Red Mix (Bioline) according to the manufacturer’s instruction. The PCR reaction primers and conditions are listed in Table S1. PCR1 reactions were set up in 25 μL with 2.5μL cDNA, while PCR2 reaction volumes were 50μL using 5 μL PCR1 product. The PCR reaction cycle was performed as 94 °C 5 min, 50 cycles of 94°C for 45 s, 60°C for 45 s and 72°C for 45 s and final extension at 72 °C for 10 min. The annealing temperature of PCR1 for lambda gene was 58°C. The amplifid bovine VH/VL genes were cloned into the human constant heavy (CH) and constant light (CL) region expression vectors in pFUSEssCHIg-hG1 and pFUSE2ss-CLIg-hL2 (Invivogen), using EcoRI/NheI and EcoRI/AvrII restriction enzymes, respectively.

Heydarchi B., Fong D.S., Gao H., Salazar-Quiroz N.A., Edwards J.M., Gonelli C.A., Grimley S., Aktepe T.E., Mackenzie C., Wales W.J., van Gils M.J., Cupo A., Rouiller I., Gooley P.R., Moore J.P., Sanders R.W., Montefiori D., Sethi A, & Purcell D.F. (2022). Broad and ultra-potent cross-clade neutralization of HIV-1 by a vaccine-induced CD4 binding site bovine antibody. Cell Reports Medicine, 3(5), 100635.

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