Quantification of protein delivery in live cells using fluorescence correlation spectroscopy

Area of Science:

• Biophysics
• Cell Biology
• Biochemistry

Background:

• Fluorescence correlation spectroscopy (FCS) is a powerful single-molecule technique.
• It measures molecular concentration and diffusion rates in vitro and in vivo.
• FCS is ideal for studying intracellular protein delivery.

Purpose of the Study:

• To detail robust FCS experimental procedures for evaluating intracellular protein delivery.
• To quantify the efficiency of cell-penetrating proteins and peptides in delivering cargo into mammalian cells.
• To assess endosomal escape efficiencies using FCS.

Main Methods:

• Preparation of fluorophore-tagged proteins for delivery studies.
• Incubation of tagged proteins with live mammalian cells and sample preparation for FCS.
• Setup and execution of FCS experiments.
• Analysis of autocorrelation curves using diffusion models and a custom MATLAB script.

Main Results:

• FCS directly quantifies the concentration and diffusion of delivered proteins.
• The method effectively evaluates the efficiency of various cell-penetrating proteins and peptides.
• Endosomal escape efficiencies can be accurately determined.

Conclusions:

• FCS provides a robust method for assessing intracellular protein delivery efficiency.
• The described procedures and analysis tools enable quantitative evaluation of protein transport and endosomal escape.
• This approach is valuable for optimizing protein delivery systems for therapeutic applications.