How to prepare the Cy3 and Cy5 labeled peptides?

  1. Peptides were synthesized using standard Fmoc solid-phase peptide synthesis.
  2. After coupling, Lys(Mtt) was selectively deprotected to expose the side-chain amine moiety. Cy3 or Cy5 were dissolved in DMF, activated, and coupled to the free amine. Cy3—and Cy5-labelled peptides were cleaved and HPLC purified according to the standard procedure.
  3. Lyophilization yielded a red powder for Cy3 and blue for Cy5.
  4. Stock solutions of non-labeled peptide (10 mM), Cy3-labelled peptide (1 mM), and Cy5-labelled peptide (1 mM) were prepared in water, aliquoted, and stored at −20 °C.
  5. Excitation wavelengths of 520 or 615 nm for Cy3 or Cy5, respectively, were used.
  6. Use emission wavelengths of 600 or 710 nm for Cy3 or Cy5, respectively.