The blocking mixture was aspirated before slides were incubated with primary antibody at 4°C overnight.
The labeled probe was added to a hybridization buffer containing 0.5 M NaCl and 5% blocking agent, and the mixture was incubated with the membrane overnight at 42°C.
Next day, the mixture was incubated with blocked protein G sepharose beads on rotation at 4 °C for 2 h.
The primary antibodies (against biotin and the protein partner to be tested) diluted in blocking solution were then added, and the mixture was incubated overnight at 4 °C.
The reaction mixture was incubated in a dry heat-block for one hour at 37°C, and immediately treated with 1 U of RQ1 DNase (Promega Corp).
The mixture was incubated at room temperature for another 1 h and the reaction was blocked by adding 3.0 μL of 2-aminoethanol.
The mixture was incubated at 65°C for 50 minutes in a heat block.
The mixture was incubated for 16 h at 45°C on the heating block and the reaction was stopped by the addition of 10% AcOH with vortexing.
The mixture was incubated for 20 min.
The mixture was incubated at 37 °C.
Then, the mixture was incubated at 40 °C for 2 minutes.
