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

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)

1. 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 use HeLa cells stably expressing EGFP-Calnexin, EGFP-CDMPR, and TfR-EGFP for illustration purposes. Apart from stable cell lines, also transiently transfe....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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