Figure 2: ECL detection of phosphoproteins on protein-blotting membranes by using the complex of Phos-tag Biotin (BTL-104) with HRP–SA. (a) Dot-blotting analysis of four non-phosphorylated proteins (bovine serum albumin, human serum albumin, carbonic anhydrase, and β-galactosidase), four phosphorylated proteins (α-casein, β-casein, ovalbumin, and pepsin), and the corresponding proteins dephosphorylated by treatment with alkaline phosphatase (APtreated). (b) Electroblotting analysis after two-dimensional electrophoresis of A431 cell lysates before (–, upper panel) and after EGF stimulation (+, lower panel). All ECL images were obtained by using the complex of Phos-tag Biotin (BTL-104) with HRP–SA, an ECL Advance Kit (as an ECL substrate reagent, GE Healthcare Bio-Sciences, Piscataway, NJ, USA), and an LAS 3000 image analyzer (Fujifilm, Tokyo, Japan). [Reprinted with permission from Ref. 19 ©(2006) The American Society for Biochemistry and Molecular Biology].