SARS-CoV-2 S1 Protein Binding Assay

Plasmids encoding the signal sequence of CD5 and a fusion of the S1 domain of the SARS-CoV S protein (residues 12–672), or the first 316 residues of that domain (12–327), with the Fc region of human IgG1 (S1–Ig and S1(327)–Ig, respectively) were transfected into 293T cells, and immunoglobulin fusion proteins were purified on protein A Sepharose beads. A total of 5 × 10⁵ 293T cells transfected with ACE2-expressing or control plasmids, or the same number of untransfected Vero E6 cells, were incubated with 15 µg ml^-1 of S1–Ig or S1(327)–Ig in a volume of 100 µl. In some cases, 15 µg ml^-1 of soluble forms of ACE1 or ACE2 (R&D Systems) was also included. Cells were washed in PBS with 0.5% BSA and 0.1% NaN₃, incubated with FITC-labelled goat anti-human IgG Fc (Sigma), and analysed by flow cytometry.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Li W., Moore M.J., Vasilieva N., Sui J., Wong S.K., Berne M.A., Somasundaran M., Sullivan J.L., Luzuriaga K., Greenough T.C., Choe H, & Farzan M. (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature, 426(6965), 450-454.

Publication 2003

293t cells Ace2 Anti igg Cells Fitc Flow cytometry Goat Human Igg1 Nan3 Plasmids Protein a sepharose Proteins Sars cov s protein Signal sequence

Corresponding Organization :

Other organizations : Brigham and Women's Hospital, Harvard University, Tufts University, University of Massachusetts Chan Medical School

Top 5 similar protocols

Protocol cited in 38 other protocols

Variable analysis

independent variables

Plasmids encoding the signal sequence of CD5 and a fusion of the S1 domain of the SARS-CoV S protein (residues 12–672), or the first 316 residues of that domain (12–327), with the Fc region of human IgG1 (S1–Ig and S1(327)–Ig, respectively)
Transfection of 293T cells with ACE2-expressing or control plasmids
Inclusion of 15 µg ml-1 of soluble forms of ACE1 or ACE2

dependent variables

Binding of S1–Ig or S1(327)–Ig to 293T cells transfected with ACE2-expressing or control plasmids, or untransfected Vero E6 cells
Binding of S1–Ig or S1(327)–Ig to cells, as measured by flow cytometry after incubation with FITC-labelled goat anti-human IgG Fc

control variables

Number of cells (5 × 10^5) used in the binding experiments
Concentration of S1–Ig or S1(327)–Ig (15 µg ml-1) used in the binding experiments
Washing of cells in PBS with 0.5% BSA and 0.1% NaN3

controls

Untransfected Vero E6 cells (negative control)
293T cells transfected with control plasmids (negative control)

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!