Evaluating the Intracellular Distribution of Virus-Derived Peptide-Modified Antibody Conjugates
Trascrizione
Begin with adhered human erythroblast cells.
Introduce virus-derived, peptide-modified antibody conjugates. These conjugates contain a nuclear localization sequence, or NLS, from the virus, and a cholic acid residue.
During incubation, the antibody conjugates interact with cell surface receptors and internalize within endosomes.
The cholic acid in the conjugate helps to escape the endosomal compartment, enabling intracellular accumulation, while the transport proteins and NLS facilitate their nuclear localization.
Introduce trypsin and EDTA-containing buffer to detach the cells and remove surface-bound conjugates.
Add media enriched with serum to inhibit trypsin activity.
Centrifuge to collect the cells; fix and permeabilize them.
Introduce far-red fluorophore-labeled secondary antibodies, binding to localized conjugates and wash.
Treat with a nuclear staining dye.
Observe under a confocal microscope and capture images at various planar depths.
After image processing, the cell shows small green fluorescent foci inside the cytoplasm and nucleus, confirming the intracellular distribution of antibody conjugates.
