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One hundred micrograms of cell lysates were incubated with anti-EGFR antibody (Santa Cruz Biotechnology) coated plates for 18 h. Biotin-EGF (Thermo Fisher Scientific) at different concentrations was added to each well and incubated at 37 °C for 3 h. After incubation with streptavidin-HRP for 1 h, the bound-EGF was detected by o-phenylenediamine dihydrochloride substrates. ELISA reader was used to detect O.D. at 490 nm. The Kd and Scatchard plot were estimated using binding data.
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