Slot binding

Proteins or peptides were applied directly to PVDF membranes using a MilliBlot-D system (Millipore). Membranes were blocked in TBS (0.05 m Tris-HCl, pH 7.5, and 0.15 m NaCl) containing 3% BSA (Sigma), incubated with ¹²⁵I-labeled protein (2 × 10⁵ cpm/ml in TBS + 3% BSA) for 3 h at room temperature or at 4 °C overnight. Membranes were then washed with TBS containing 0.05% Tween 20, and binding was visualized using the Fuji FLA-3000 imaging system.