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. The 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-labelled 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. Use excitation wavelengths of 520 or 615 nm for Cy3 or Cy5, respectively.
  6. Use emission wavelengths of 600 or 710 nm for Cy3 or Cy5, respectively.