The rCstegu20.6 proteins were subjected to 12% SDS-PAGE and subsequently transferred to PVDF membranes (Millipore, 0.45 µm, IPVH00010, Molsheim, France). Non-specific binding was blocked by incubation in blocking buffer (5% skim milk, 20 mM Tris, 500 mM NaCl, 0.05% v/v Tween 20, pH 7.4) at room temperature. The membranes were reacted with HRP conjugated anti-polyhistidine antibody (mouse, polyclonal, Abcam, ab49781, 1:2000 dilution) for 1 h. After washing with TBST, the immunoreactive bands were visualized on the membrane using 4-choloro-1-naphthol solution (Sigma, C6788, St. Louis, MO, USA).