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oai:figshare.com:article/31368386 2026-09-17T19:04:21Z category_1 category_4 category_915 category_7 category_12 category_14 category_873 category_734 category_133 portal_63 item_type_2 month_year_09_2026

Cell-Penetrating Peptides and Supercharged Proteins: A Comprehensive Protocol from Isolation to Cellular Uptake Alexander V. Beribisky (23217536) Victoria Sarne (23217539) Anna Huber (3459476) Markus Hengstschläger (2120059) Franco Laccone (730656) Hannes Steinkellner (23217542) Biophysics Biochemistry Physical Sciences not elsewhere classified Medicine Cell Biology Molecular Biology Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified Plant Biology dynamic light scattering reproducible pipeline adaptable binding protein 2 protocol integrates advances imaging flow cytometry cell fluorescence microscopy mecp2 sequence probing cellular uptake analysis sps using methyl cellular uptake cell validate transducing cpp based protein constructs cellular uptake cell imaging structural analysis reproducible workflows protein biochemistry based readouts replicable protocol content imaging wide range therapeutic interventions therapeutic development term stability supercharged proteins principle model practical framework penetrating peptides like motifs like motif internalized signal fusion proteins expanding applications downstream mechanistic basic research additional assays Cell-penetrating peptides (CPPs) and supercharged proteins (SPs) enable efficient intracellular delivery of macromolecules, with expanding applications in basic research and in therapeutic development. Despite their potential, reproducible workflows for isolation, biochemical characterization, and quantitative uptake analysis remain limited. Here, we present a comprehensive and replicable protocol for the isolation, characterization, and cellular uptake analysis of CPP-fusion proteins (CPP-FPs) and SPs using methyl-CpG-binding protein 2 (MeCP2) constructs as a proof-of-principle model. This workflow combines native protein purification with dynamic light scattering (DLS)-based buffer optimization. Cellular uptake is then assessed and quantified under live-cell conditions using high-content imaging and imaging flow cytometry, with additional assays to probe endocytic trafficking routes, identify CPP-like motifs in SPs, and validate transducing CPP-FP/SP functionality. The protein isolation and DLS-guided buffer screen yield samples with long-term stability. Live-cell fluorescence microscopy and imaging flow cytometry enable discrimination between membrane-bound and internalized signal, providing higher accuracy compared to plate-based readouts. MeCP2 sequence probing has revealed the presence of a CPP-like motif that is critical to its internalization. Finally, validation assays clearly demonstrated CPP-FP/SP activity. This protocol integrates advances in protein biochemistry, structural analysis, and live-cell imaging into a reproducible pipeline adaptable to a wide range of CPP- and SP-based protein constructs and provides a practical framework for downstream mechanistic and therapeutic interventions. 2026-03-02T00:00:00Z Dataset Media 10.1021/acs.molpharmaceut.5c01560.s001 https://figshare.com/articles/media/Cell-Penetrating_Peptides_and_Supercharged_Proteins_A_Comprehensive_Protocol_from_Isolation_to_Cellular_Uptake/31368386 CC BY-NC 4.0