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
Figure 1
(A) Beads coated with interacting partners (A and B) are expected to aggregate when mixed. (B) SVA-beads coated with biotin-labelled double-stranded DNA are prepared with one population displaying a sticky end (DNA-F) and the other with a complementary sticky end (DNA-F′). Interactions between the complementary strands are expected to cause aggregation of the beads. (C) SVA beads displaying either DNA-F or DNA-F′ were prepared. Each DNA can only weakly self-dimerize with a predicted KD of ∼36 mM, while a mixture of complementary DNA can form dimers with a predicted KD of ∼63 fM (calculated using OligoAnalyzer 3.1, Integrated DNA Technologies). Differential interference contrast (DIC) microscopy was used to measure aggregation after incubation (25 µL, 30 min) of each population of beads alone (10 pmol of SVA sites and 20 pmol biotin-DNA) or after mixing (50∶50 ratio). The scale bar (20 µm) applies to all three images. (D) Summary results show the aggregation ratios (see Methods) as means ± SEM, from 3 independent experiments with 5 fields analyzed in each. *P = 0.017.