Fig. 2

Purification and characterization of the five anti-EGFR sdAbs. a SDS-PAGE was performed to examine the five anti-EGFR sdAbs (aEG1B4, aEG2C7, aEG2E12, aEG4D9 and aEG6B2). Left lane, protein marker; lane 1, uninduced total bacterial protein; lane 2, induced total bacterial protein; lane 3, insoluble fraction after bacterial breakage; lane 4, soluble fraction after bacterial breakage; lane 5, flow-through fraction; lane 6, wash buffer; lane 7-11, fractions collected from the protein purification column. The sdAb positions were marked by the arrows. b The specific binding of the five purified anti-EGFR sdAbs to the EGFR protein fragment was evaluated with ELISA. EGFR protein fragment and the other seven unrelated proteins (VEGF, EndoF1, CampH, HER2, BMP2, FGF21 and CXCR4) as negative controls were included. c Western blot analysis was performed with the five purified anti-EGFR sdAbs against the EGFR complete extracellular fragment purchased commercially (lane 2) and PBS as a control (lane 1). The specific binding of the five purified anti-EGFR sdAbs to cancer cells was examined by flow cytometric analysis (d–h). The three human cancer cell lines A549 (d), MCF-7 (e) and DU145 (f) were tested. The two cell lines 293T (g) and 3T3 (h) were included as negative controls. Black curves showed the background staining with an isotype control primary antibody or no primary antibody. Red curves showed the staining with anti-EGFR antibody purchased commercially as a positive control, the five purified anti-EGFR sdAbs (aEG1B4, aEG2C7, aEG2E12, aEG4D9 and aEG6B2), or the two purified sdAbs (aVE201 and aHer2-13C1) as negative controls