Work

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Year: 2014

Type: article

Abstract: Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Source: Journal of Visualized Experiments

DOI: 10.3791/51762

Authors: Michelle R. Emond, James D. Jontes

Institution: The Ohio State University

Language: English

Topic: Cell Adhesion Molecules Research

Subfield: Immunology and Allergy

Field: Medicine

Domain: Health Sciences

Open Access status: bronze

Funders: National Science Foundation, National Institutes of Health, National Institute of Mental Health

Awards: Mechanism of differential adhesion by protocadherin-cadherin complexes, The role of Protocadherin-19 in brain morphogenesis