If a selected sequence binds the target in the context of intact phage, but not as a synthetic peptide, it is possible that the selected sequence requires additional elements from the adjacent spacer sequence for binding. Bear in mind that, while the N-terminus of the selected peptide sequence was free during panning, the C-terminus was fused to the phage. Furthermore, the C-terminal residue of the selected sequence did NOT have a free negatively-charged carboxylate during panning, so a simple synthetic peptide with a free carboxy terminus will introduce a negatively charged group at a position occupied by a neutral peptide bond during panning, which may completely abolish binding. When designing synthetic peptides corresponding to selected sequences, we recommend adding the spacer sequence Gly-Gly-Gly-Ser to the C-terminus, and if possible, amidating the C- terminal carboxylate to block the negative charge. For chemical conjugation of the peptide to a reporter enzyme, the C-terminal serine can be replaced with cysteine (if there are no other cysteines present in the sequence). The resulting peptide thiol can be easily coupled to maleimide-activated HRP or alkaline phosphatase (both available from Pierce).