Her2 Receptor Quantification by ELISA

Binding of affinity module alone and of affinity-module-dCK fusion proteins to ectodomain IV of Her2 was detected by performing a protein ELISA as described before [32 (link)]. Briefly, 20 nM mouse anti-His antibody (Invitrogen) was used to capture 20 nM of recombinant human ErbB2/Her2 Fc Chimera (R&D Systems) onto Nunc polystyrene microtiter plate wells (Thermo Fisher Scientific). Fifty nM of the affinity module alone or of the affinity-module-dCK fusion protein were added to the wells, and binding detected with an anti-Myc antibody (9E10) conjugated to biotin (Santa Cruz Biotechnology) and streptavidin-conjugated to horseradish peroxidase (HRP; Abcam). The human Her2 ELISA Kit (Invitrogen) was used to determine the number of Her 2 receptors in cell lines. Briefly, 10⁷ cells were lysed in cell extraction buffer (Invitrogen), diluted to a volume of 100 μL/well, and the copy number of Her2 receptor/ cell calculated according to the manufacturer’s protocol.