Monitoring the Arrival of GFP-Tagged Surface Proteins to the Trans-Golgi Network Using Functionalized Nanobodies
Trascrizione
Start with sulfate-deprived cancer cells expressing a GFP-tagged surface protein.
Add radiolabeled sulfate and modified anti-GFP nanobodies — a single-chain antibody carrying a tyrosine-sulfation motif, and incubate.
Sulfate-deprived cells internalize radiolabeled sulfates, incorporating them into cellular components.
The anti-GFP nanobody binds to the GFP-tagged surface protein and gets endocytosed into a vesicle. This vesicle transports the nanobody-surface protein complex along specific intracellular pathways, including the trans-Golgi network, or TGN.
Within the TGN, the sulfotransferase in the presence of a substrate transfers a radioactive sulfate to specific tyrosine residues on the nanobody, thereby radiolabeling it.
Wash the cells. Next, lyse the cells.
Centrifuge the lysate to remove large cellular debris. Isolate the nanobodies using a nickel bead-based purification method.
Load the isolated fraction on an SDS-PAGE gel to separate the radiolabeled nanobody into distinct bands.
Subject the gel to autoradiography.
The resulting autoradiograph reveals darkened regions, indicating the presence of the radiolabeled nanobody.
