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.
1. Uptake of Functionalized Nanobodies by Cultured Cells (for Sulfation Analysis)
The authors have nothing to disclose.
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 |