ABSTRACT Labeling peptides with fluorophores remains the dominant approach for assessing their cellular uptake, yet this process is time‐intensive, costly, and can modify peptide structure and biological behavior. Here a label‐free fluorescence‐based screening method is presented that exploits the environmental sensitivity of 1‐anilino‐8‐naphthalene sulfonate (ANS) to monitor peptide–membrane interactions in real time. ANS shows negligible emission in water but undergoes a characteristic blue shift and intensity enhancement upon association with hydrophobic regions. These features were used to distinguish penetrating from non‐penetrating peptides in both plant protoplasts and mammalian HEK 293 T cells. Classical cationic cell‐penetrating peptides (CPPs), poly‐arginine (R9) and TAT (49–57), produced distinct ANS responses within minutes, while the non‐penetrating mutant mTAT showed no detectable effect. The ANS‐based assay provides a cost‐efficient, label‐free, and high‐throughput tool for screening native peptides and offers new insight into the hydrophobic transitions that accompany peptide internalization.
Vivek Kumar (Fri,) studied this question.