Hi everyone,
FLAG was fused with my protein at N terminal. I performed immunostaining and could see fluorescence. However, when I checked its expression in western blotting, I could never detect my protein using FLAG antibodies (sigma, F1804, cell signaling, 2368). Whereas, when I used custom raised antibody against protein C terminal, I could see very strong band corresponding to correct size of protein(using the same protein aliquots as above western blotting where Flag anitbodies were used). Now, it is obvious that FLAG antibodies cannnot recongnize FLAG in western blotting. Does anyone know the reason? What can I do to solve this problem? Which antibody would you recommend to me? Thanks for your input!
Best!
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