Document Type

Article

Department

​Chemistry​

Publication Date

9-2018

Abstract

Studies of live cells often require loading of exogenous molecules through the cell membrane; however, effects of loading method on experimental results are poorly understood. Therefore, in this work, we compared three methods for loading a fluorescently labeled peptide into cells of the model organism Dictyostelium discoideum. We optimized loading by pinocytosis, electroporation, and myristoylation to maximize cell viability and characterized loading efficiency, localization, and uniformity. We also determined how the loading method affected measurements of enzyme activity on the peptide substrate reporter using capillary electrophoresis. Loading method had a strong effect on the stability and phosphorylation of the peptide. The half-life of the intact peptide in cells was 19 ± 2, 53 ± 15, and 12 ± 1 min, for pinocytosis, electroporation, and myristoylation, respectively. The peptide was phosphorylated only in cells loaded by electroporation. Fluorescence microscopy suggested that the differences between methods were likely due to differences in peptide localization.

Comments

Author's accepted manuscript. Published version available: https://doi.org/10.1021/acs.analchem.8b02111

Publication Title

Analytical Chemistry

Volume

90

Issue

19

First Page

11344

Last Page

11350

DOI

10.1021/acs.analchem.8b02111

Download

Included in

Chemistry Commons

Share

COinS