A colorimetric bead-binding assay for detection of intermolecular interactions.
We have developed a new technique that rapidly and reproducibly allows direct visualization of molecular interactions, including receptor-ligand binding. The technique can be easily applied to examine binding between proteins and glycoproteins, or proteins and glycolipids, including gangliosides. In this novel bead-binding assay, the suspected 'ligand' molecule is bound to 0.2- or 1.0- micro m colored FluoSphere beads. These coated beads are then mixed directly with 150- micro m Sepharose or 50- to 75- micro m agarose beads coated with the second 'target' molecule. Binding between molecules is easily detected by immunofluoresence microscopy as colored rosettes or aggregations formed by clustering of the smaller fluorescent beads around the larger non-fluorescent bead. The validity of this technique for glycolipid binding to protein was verified through demonstration of the known interaction between the beta subunit of cholera toxin with ganglioside GM1. The bead-binding technique facilitated the novel observations of interaction between ganglioside GT1b with the alpha5 subunit of alpha5beta1 integrin and the interaction of GM3 with the epidermal growth factor receptor. A modification of this technique, in which the coated beads are bound to protein fixed on plates, allows a quantifiable colorimetric assay of interaction. This versatile and rapid technique will have widespread applications for in vitro systems and may also be useful for in vivo analysis of binding to cell surface receptor molecules.
D000818:Animals; D002772:Cholera Toxin / Q000378:metabolism; D003124:Colorimetry / Q000379:methods*; D066246:ErbB Receptors / Q000378:metabolism; D005456:Fluorescent Dyes; D005677:G(M1) Ganglioside / Q000378:metabolism; D005679:G(M3) Ganglioside / Q000378:metabolism; D005732:Gangliosides / Q000378:metabolism; D066298:In Vitro Techniques; D039081:Integrin alpha5beta1 / Q000378:metabolism; D008024:Ligands; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*
D005456:Fluorescent Dyes; D005679:G(M3) Ganglioside; D005732:Gangliosides; D039081:Integrin alpha5beta1; D008024:Ligands; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D005677:G(M1) Ganglioside; C016481:trisialoganglioside GT1; D002772:Cholera Toxin; D066246:ErbB Receptors