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 4
The NT of IP3R1 causes aggregation of beads.
(A) DIC images of beads prepared as indicated. (B) Summary results show means ± SEM, n = 3. *P = 0.012 relative to SVA beads. (C) Western blot, probed using HRP-conjugated streptavidin, shows equivalent fractions of the input (20 pmol of native or denatured NT-biotin, lane 1), the supernatant (lane 2), wash (lane 3) and NT-beads treated (85°C, 10 min) to release bound protein (lane 4). Lane 5 shows the biotinylated protein ladder. The ∼70-kDa bands correspond to NT-biotin. The blot is representative of two similar analyses. (D) DIC images of NT-biotin-coated SVA-beads with or without 10 µM IP3. (E) Summary results show means ± SEM, n = 5. P = 0.40. Scale bars (A and D) represent 20 µm.