After the transference (in approximately one hour at 0.70 steady amperes), the nitrocellulose membrane was immersed in blocking solution (casein - Non-fat Dry Milk, Biorad) for one hour.
They were washed four times for 15 minutes each time, at room temperature, with TBS-T [Tris-HCl 0.01mol/L, NaCl 1.5mmol/L, Tween-20 0.1% (v/v), pH 7.6].