Monitoring the Arrival of GFP-Tagged Surface Proteins to the Trans-Golgi Network Using Functionalized Nanobodies

Published: January 31, 2024

Abstract

Source: Buser, D. P., et al. Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells. J. Vis. Exp. (2019).

This video demonstrates a method to study the uptake and retrograde transport of functionalized nanobodies to understand their intracellular trafficking pathways. By introducing radiolabelled sulfate and utilizing isolation and detection techniques, this method enables the study of endocytic uptake and trans-Golgi network arrival of cargo proteins.

Protocol

1. Uptake of Functionalized Nanobodies by Cultured Cells (for Sulfation Analysis) In a cell culture hood, seed ~400,000 to 500,000 HeLa cells stably expressing a GFP-tagged reporter protein in 35 mm dishes or on 6-well clusters with complete medium containing antibiotics (Dulbecco's Modified Eagle medium, DMEM, supplemented with 10% fetal calf serum (FCS), 100 units/mL streptomycin, 2 mM l-glutamine, and 1.5 µg/mL puromycin). NOTE: As mentioned above, we here …

Disclosures

The authors have nothing to disclose.

Materials

Anti-GFP antibody Sigma-Aldrich 1.18145E+11 Product is distributed by SigmaAldrich, but manufactured by Roche
Dulbecco's modified Eagle's medium (DMEM) Sigma-Aldrich
Fetal calf serum (FCS) Biowest
Sulfur-35 as sodium sulfate Hartmann Analytics ARS0105 Product contains 5 mCi
6-well plates TPP 92406
Paraformaldehyde (PFA) Applichem A3813
Fluoromount-G Southern Biotech 0100-01
Mini-Protean TGX gels, 4-20%, 15- well Bio-Rad 456-1096

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Cite This Article
Monitoring the Arrival of GFP-Tagged Surface Proteins to the Trans-Golgi Network Using Functionalized Nanobodies. J. Vis. Exp. (Pending Publication), e21900, doi: (2024).

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