The membrane was soaked in PBS-T buffer (Na2HPO4 5.75 g, NaH2-
PO4?2H2O 1.48 g, NaCl 2.92 g, and Tween-20 0.5 mL) added with 5% skim milk for 1 h. After being washed three times in PBS-T buffer, the membrane was incubated for 2 h with His-tag antibody.
Membrane washing was repeated three times with PBS-T buffer and then bands were visualized by the reaction ECL Western blotting detection reagents (GE Healthcare Biosciences, Pittsburgh, PA,USA) on the membrane.