Sema3G Protein Expression and Purification

The Sema3G expression vector has been described previously [12 (link)]. The vector was transfected into 293T cells using Lipofectamine LTX (Thermo Fisher Scientific). Cells were cultured in DMEM supplemented with ultra-low IgG FBS (Gibco). The culture supernatant was collected and dialyzed using Amicon Ultra 100 K (Merck). The dialyzed supernatant was further purified with a Protein G column (Cytiva), and the buffer was exchanged with PBS. Sema3G production was confirmed by Coomassie blue staining and western blotting.

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Shoda J., Tanaka S., Etori K., Hattori K., Kasuya T., Ikeda K., Maezawa Y., Suto A., Suzuki K., Nakamura J., Maezawa Y., Takemoto M., Betsholtz C., Yokote K., Ohtori S, & Nakajima H. (2022). Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium. Arthritis Research & Therapy, 24, 134.

Publication 2022

Lipofectamine LTX ultra-low IgG FBS Amicon Ultra 100 K Protein G column

293t cells A protein Buffer Cells Coomassie blue Lipofectamine Protein g Vector

Corresponding Organization : Chiba University

Other organizations : International University of Health and Welfare, Uppsala University

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Variable analysis

independent variables

Transfection of Sema3G expression vector into 293T cells using Lipofectamine LTX

dependent variables

Sema3G production

control variables

Culturing 293T cells in DMEM supplemented with ultra-low IgG FBS
Dialysis of culture supernatant using Amicon Ultra 100 K
Purification of dialyzed supernatant with a Protein G column
Buffer exchange with PBS

controls

Positive control: Coomassie blue staining and western blotting to confirm Sema3G production

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