For example, cell penetrating peptides (CPPs) modified with FITC and photoactivatable probes have been used to track binding patterns and dynamic behavior over time. Unlike proteins, these peptides localize to specific targets on actin and are less prone to protein aggregation, making them ideal for in vitro tracking (Pan 2014). Similarly, FITC-labeled CPPs have been used to image intracellular compartments of cells with low risk of cytotoxicity (Kirkham 2015).
For example, high resolution near-infrared fluorophores have been used as fibrin imaging-agents for deep vein thrombosis (DVT). Using Cy7 labeled fibrin-targeting peptides, CT scanning and confocal microscopy were used to differentiate between acute and subacute murine DVT (Hara 2012). Similar techniques have also been used for detecting apoptosis as a symptom for glaucoma. Fluorophore-labeled peptides that are activated by caspases can be imaged in vivo to track glaucoma progression (Qiu 2014).
In these studies, a donor molecule, such as Abz (Marcondes 2015) or Lucifer Yellow (Rossé 2000) is covalently attached to the C-terminus, while acceptor molecules (ex. Dabsyl, DNP) are coupled to the N-terminus. This strategy is commonly employed with peptides synthesized using a solid-phase approach, and peptides are left conjugated to the bead. If the peptides are not targeted by protease activity, both the donor and acceptor will be present, and the bead will appear non-fluorescent. If the peptides are cleaved, the peptide will no longer be quenched by FRET and the beads will fluoresce. Since protease studies are most effective when FRET is combined with shorter peptides, micro-scale peptide libraries are ideal choices for these experiments.