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

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.

In a cell culture hood, seed between 400,000 and 500,000 HeLa cells that stably express a GFP-tagged reporter protein into either 35mm dishes or 6-well clusters, with complete medium containing antibiotics. Incubate the cells at 37 degrees Celsius with 7.5% carbon dioxide overnight. The next day, remove the complete medium, and wash the cells twice with 1X PBS at room temperature. Starve the cells with sulfate-free medium for 1 hour at 37 degrees Celsius with 7.5% carbon dioxide.

Meanwhile, in a ventilated hood or bench designated for radioactivity work, prepare sulfate labeling medium containing 0.5 millicuries per milliliter sodium sulfate S35 in sulfate-free medium. Next, mix VHH double-tier-sulf in 1X PBS with sulfate labeling medium to a final concentration of 2 micrograms per milliliter. When the incubation is complete, replace the sulfate-free media with 0.7 milliliters of the prepared medium mixture, containing sodium sulfate S35 and VHH double-tier-sulf. Incubate the cells for 1 hour at 37 degrees Celsius in a 7.5% carbon dioxide incubator, designed for work with radioactivity.

After this, remove the labeling medium, and wash the cells two to three times with ice-cold 1X PBS on either a cooling plate or on ice. Add 1 milliliter of lysis buffer, supplemented with two millimolar PMSF and 1X protease inhibitor cocktail. Incubate the cells for 10 to 15 minutes on a rocking platform at 4 degrees Celsius. Then, scrape and transfer the lysate to a new 1.5-milliliter tube, vortex the lysate, and place it on an end-over-end rotator at 4 degrees Celsius for 10 to 15 minutes. Centrifuge at 10,000 g and at 4 degrees Celsius for 10 minutes to prepare a post-nuclear lysate. Transfer the post-nuclear lysate into a 1.5-milliliter tube containing prepared nickel beads, and incubate on an end-over-end shaker at 4 degrees Celsius for 1 hour to isolate the nanobodies.

After this, wash the beads three times with either 1X PBS or lysis buffer by gently pelleting at 1000 g for 1 minute. Carefully remove all of the washing buffer from the beads, and add 50 microliters of 2X sample buffer. Boil at 95 degrees Celsius for 5 minutes. Then, load the boiled beads on a midi 12.5% SDS-PAGE gel, and run according to the standard PAGE protocol until the reference dye has reached the end of the separating gel. Fix the separating gel in approximately 30 milliliters of fixation buffer for 1 hour at room temperature. Wash the gel three times with approximately 50 milliliters of deionized water, and then dry the gel, as outlined in the text protocol. When the drying is complete, use a phosphor screen imager to image autoradiograph the gel.