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 …
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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 |
