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 transfected HeLa can be used.
2. Incubate the cells O/N at 37 °C in a 7.5% CO₂ incubator to proliferate.
   NOTE: Cells should have a confluency of ~80% the next day.
3. Remove the complete medium, wash 2 times with 1x PBS at RT, and starve the cells with sulfate-free medium (SFM) for 1 h at 37 °C in a 7.5% CO₂ incubator.
   NOTE: SFM is prepared by adding 10 mL of MEM amino acids (50x) solution, 5 mL of L-glutamine (200 mM), 5 mL of vitamin solution (100x), and 900 µL of CaCl₂·2H₂O to 500 mL of Eagle's balanced salts. Pass through a 0.22 µm filter and aliquot.
4. In a ventilated hood or bench designated for radioactivity work, prepare a sulfate labeling medium containing 0.5 mCi/mL [³⁵S] sulfate as a sodium salt in SFM.
   CAUTION: Carefully handle all solutions containing radioactivity. Only work in designated hoods or benches. All material (tips, tubes, plates, etc.) or wash buffers in contact with radioactivity have to be collected and disposed of separately. Do this for all steps below (steps 1.5-1.18) as well.
5. Add VHH-2xTS in 1x PBS into sulfate labeling medium to a final concentration of 2 µg/mL.
   NOTE: As a control, also include VHH-std or another nanobody devoid of TS sites to demonstrate specific labeling.
6. Replace SFM with 0.7 mL of sulfate labeling medium containing 0.5 mCi/mL [35S]sulfate and 2 µg/mL VHH-2xTS and incubate the cells for 1 h at 37 °C in a 7.5% CO₂ incubator designated for work with radioactivity.
   NOTE: Depending on the planned experiment, the time of nanobody sulfation needs to be adjusted. In addition, to determine TGN arrival kinetics, labeling is performed for different times (e.g., for 10 min, 20 min, 30 min, etc.).
7. Remove the labeling medium and wash the cells 2-3 times with ice-cold 1x PBS on a cooling plate or ice.
8. Add 1 mL of lysis buffer (PBS containing 1% Triton X-100 and 0.5 % deoxycholate) supplemented with 2 mM PMSF and 1x protease inhibitor cocktail and incubate the cells for 10-15 min on a rocking platform at 4 °C.
9. Scrape and transfer the lysate into a 1.5 mL tube, vortex the lysate, and place for 10-15 min on an end-over-end rotator at 4 °C.
10. Prepare a postnuclear lysate by centrifugation at 10,000 x g for 10 min at 4 °C.
11. Using a new 1.5 mL tube, prepare 20-30 µL of a slurry of Nickel beads in a 1.5 mL tube and wash once with 1x PBS by gently pelleting them at 1,000 x g for 1 min.
    NOTE: Instead of Nickel beads, streptavidin beads (if the nanobody is biotinylated) or protein A beads with an IgG against an epitope of the nanobody (T7- or HA-tag) can be used.
12. Transfer the postnuclear lysate into a 1.5 mL tube containing Nickel beads and incubate for 1 h at 4 °C on an end-over-end rotator to isolate the nanobodies. Remove an aliquot (50-100 µL) of the postnuclear lysate and boil in SDS sample buffer for subsequent immunoblot analysis of total cell-associated nanobody, GFP reporter, and loading control.
13. Wash the beads 3 times with 1x PBS or lysis buffer by gently pelleting at 1,000 x g for 1 min.
    NOTE: For more stringent washing of beads, 20 mM imidazole can be included in PBS or lysis buffer.
14. Carefully remove all washing buffer from the beads, add 50 µL of 2x sample buffer, and boil for 5 min at 95 °C.
15. Load both boiled beads on a midi 12.5% SDS-PAGE gel and run according to the standard PAGE protocol until the reference dye (e.g., bromophenol blue) has reached the end of the separating gel.
    NOTE: Immunoblot analysis of total cell-associated nanobody, GFP reporter, and loading control can also be performed using a mini 12.5% SDS-PAGE or precast gradient gel.
16. Fix the separating gel in ~30 mL of fixation buffer (10% acetic acid and 45% methanol in ddH₂O) for 1 h at RT, wash the gel three times with ~50 mL of deionized water, and prepare for gel drying.
17. Drying SDS-PAGE Gels.​
    1. Carefully place the fixed gel on stretched cling film and cover it with precut filter paper.
    2. Place the gel with the filter paper down on top of the drying platform, place the vacuum cover, and switch on the vacuum pump for gel drying. Dry the gel for 3-5 h.
    3. Remove the cling film and place the dried gel into an autoradiography cassette equipped with a Phosphor Screen.
18. Image autoradiograph using a Phosphor Screen Imager. Follow the instructions of the manufacturer.
    NOTE: Depending on the strength of the signal, dried gels need to be exposed longer with Phosphor screens.