I want to check peptide specificity with EGFR. I think that peptide should internalize through EGFR in EGFR positive cell lines. I took A549 and Cal 33 cell line, both are EGFR positive cell line. I got also results from FACS experiment that peptide internalize in both cell lines in a good percentage. So, I want to check specificity of the peptide by blocking EGFR with EGF. I'm facing some problem repeatedly in blocking experiment, in my case I didn't see any peptide internalization in both well with EGF and without EGF. How is it possible? The same experiment I did only with peptide for concentration and time assay. Every time I got good signal in fluorescence microscopy. But when I did the same experiment in presence of EGF I didn't get any GFP signal for peptide. In presence of EGF its expectable that I will not get any signal but where only I add the peptide there also I didn't get any signal. This issue happening repeatedly. What's the solution if someone knows please response to my question. Thank you very much.