title: A bead aggregation assay for detection of low-affinity protein-protein interactions reveals interactions between N-terminal domains of inositol 1,4,5-trisphosphate receptors.
abstract: Interactions between proteins are a hallmark of all cellular activities. Such interactions often occur with low affinity, a feature that allows them to be rapidly reversible, but it makes them difficult to detect using conventional methods such as yeast 2-hybrid analyses, co-immunoprecipitation or analytical ultracentrifugation. We developed a simple and economical bead aggregation assay to study low-affinity interactions between proteins. By coating beads with interacting proteins, the weak interactions between many proteins are sufficient to allow stable aggregation of beads, an avidity effect. The aggregation is easily measured to allow quantification of protein-protein interactions under a variety of controlled conditions. We use this assay to demonstrate low-affinity interactions between the N-terminal domains of an intracellular Ca(2+) channel, the type 1 inositol 1,4,5-trisphosphate receptor. This simple bead aggregation assay may have widespread application in the study of low-affinity interactions between macromolecules.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Alap P
authors.initials: AP
authors.lastname: Chavda
authors.forename: David L
authors.initials: DL
authors.lastname: Prole
authors.forename: Colin W
authors.initials: CW
authors.lastname: Taylor
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D047468:Immunoprecipitation; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000191:economics / Q000379:methods*; D051381:Rats
chemical_list: D055767:Immobilized Proteins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015544:Inositol 1,4,5-Trisphosphate
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