C3 VHH has a high affinity and specificity to Her2. (A) Her2-Fc was expressed and purified from HEK293f cells efficiently. Coomassie blue staining of the SDS-PAGE showed that C3-Fc was approximately 45 kDa. M indicates the molecular weight; E1, E2, and E3 refer to elution 1, 2, and 3 from the protein A affinity chromatograph. (B) Immunofluorescence analysis of Her2 binding. CHO and SKBR3 cells were fixed and stained with DAPI (nucleus staining), C3-Fc (left) or trastuzumab (right) were the primary antibodies, and then, Alex488-conjugated goat anti-human IgG1 was the secondary antibody. (C) Flow cytometry analysis of Her2-Fc proteins. The tumor cells CHO, MCF7, LS174T, and SKOV3 were studied. The gray area (blank) indicates cells with no staining; the dashed line indicates cells with human IgG1 and Alex488-conjugated goat anti-human IgG1 staining; the solid line indicates C3-Fc staining, then with Alex488-conjugated goat anti-human IgG1 staining; the dotted line indicates trastuzumab staining, then with Alex488-conjugated goat anti-human IgG1 staining.