2.5. Probing Protein:Peptide Interactions
Membranes were pre-wetted by rinsing several times with ethanol followed by 3 × 5 min washes with PBST buffer (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, 0.1% Tween 20, pH 7.4). They were subsequently blocked with 5% BSA in PBST buffer for 8 h at room temperature in order to reduce non-specific binding. After 2 washes with PBST buffer (5 min each) followed by a single wash with PBS buffer (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4) for 5 min, His6-tagged BRDs were added at a final concentration of 1 µM before an overnight incubation in PBS buffer at 4 °C. Each membrane was washed 3 times in PBST buffer to remove any unbound protein, blocked for 1 h with 5% BSA in PBST buffer, and washed again 3 × 5 min with PBST buffer to remove the excess of BSA. His-tag® Antibody HPR conjugated (Novagen, distributed by Merck-Millipore, #71841) was added in 1% BSA/PBST solution at a dilution of 1:3000. After 1 h incubation, membranes were washed 3 × 20 min in PBST buffer to remove any excess antibody. We found the large number of washing steps necessary in order to avoid low signal to background ratio. All incubation and washing steps were performed using a PMR-30 Compact Fixed-Angle Platform Rocker which was set to 30 oscillations per minute.