Materials and Reagents

15 ml conical tube
Beckman tube (Beckman Coulter, catalog number: 343776 )
Pipette tip with a diameter of 0.6 mm (e.g., Gel-Saver^® II, STARLAB, catalog number: I1022-0600 )
Spin Desalting Column, 2 ml, 10 columns, for 200-700 μl samples, 7,000 MWCO (Zeba^TM, catalog number: 89882 )
Tissue of choice (e.g., Drosophila embryos)
GFP VHH, recombinant binding protein (Chromotek, catalog number: gt-250 )
1x Phosphate-buffered Saline (PBS): 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2
Sulfo-NHS-SS-Biotin (Thermo Scientific, catalog number: 21945 )
Anhydrous dimethylformamide (DMF) (Sigma-Aldrich, catalog number: 227056 )
Buffer of choice (e.g., 1x BRB80: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, pH 6.9 at 4 °C)
IGEPAL (Sigma, catalog number: I8896 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma, catalog number: 10837091001/PMSF-RO )
Pi (cOmplete Mini protease inhibitor) (Roche, catalog number: 11836170001 )Working concentration: 1 tablet per 10 ml extraction solution
PPi (PhosSTOP phosphatase inhibitor) (Roche, catalog number: 4906837001 )Working concentration: 1 tablet per 10 ml extraction solution
High Capacity Streptavidin Agarose Resin (Pierce, catalog number: 20349 )
Dithiothreitol (DTT) (Thermo scientific, catalog number: R0861 )
Biotinylated antibody/nanobody (Chromotek, catalog number: gt-250 )
Liquid nitrogen

Equipment

Optima MAX-TL ultracentrifuge with TLA-120.1 rotor (Beckman Coulter)
Microcentrifuge
Rocking platform shaker
Rotating disc mixer
Dounce homogenizer
-80 °C freezer

Procedure

Preparation of GFP-TRAP-Sulfo beads
Desalt/Buffer Exchange of the nanobody (e.g., GFP-binding-protein)
Note: The nanobody must be completely desalted into 1x PBS before being modified with Sulfo-NHS-SS-Biotin.

Zeba^TM Spin desalting columns are used according to manufacturer's instructions.
1. Remove column’s bottom closure, loosen the cap, and place into a 15 ml conical tube.
2. Centrifuge at 1,000 x g for 2 min in a benchtop microfuge. Discard the flow-through (to remove storage buffer).
3. Place a mark on the side of the column where the compacted resin is slanted upward. Place column in the microcentrifuge with the mark facing outward in all subsequent centrifugation steps.
4. Equilibrate the Zeba column with 1 ml 1x PBS. Centrifuge at 1,000 x g for 2 min and discard the flow-through.
5. Repeat Step A4 two more times.
6. Insert the equilibrated column in a new conical tube. Apply 200 μl of nanobody (1 mg/ml) directly on the filter.
7. Centrifuge at 1,000 x g for 2 min and collect the flow through (nanobody).
Biotin-labelling Reaction
1. Allow vial of Sulfo-NHS-SS-Biotin to equilibrate to ambient temperature before opening.
2. Immediately before use, dissolve the Sulfo-NHS-SS-Biotin in DMF to give a 8 mM solution.
3. Add 86.1 μl of Sulfo-NHS-SS-Biotin solution to 200 μl of nanobody (1 mg/ml).
4. Mix immediately by inversion.
5. Incubate on ice for 2 h.
6. Stop the reaction by removing the excess biotin reagent using a fresh Zeba spin column (i.e., repeat Steps A1-A7).
Linking the Biotinylated antibody to Streptavidin Agarose beads
1. Mix the streptavidin agarose resin to ensure an even suspension.
2. Wash 40 μl of bead slurry (20 μl packed beads) by resuspension in 1x PBS, pelleting the beads at 2,500 x g for 2 min in a microcentrifuge. Repeat three more times.
3. Add biotinylated nanobody solution (from B6) to washed streptavidin agarose beads and allow to bind to beads using either a rocking platform shaker or a rotating disc mixer for 1 h at 4 °C.
4. Remove any unbound antibody by washing three times as described above (C2) but using the buffer of choice (e.g., BRB80 + 0.1% IGEPAL) instead of PBS.
5. After removing the supernatant from the last wash, resuspend the beads in the buffer of choice, to maintain a 50% (40 μl) bead slurry. Store at 4 °C.
Note: Cl-AP beads can be stored at 4 °C for at least 3 months with no loss of activity.
Homogenization of tissue and preparation of the High-speed Supernatant (HSS)
Drosophila embryos expressing the GFP-fusion protein of interest are collected (batches of 0- to 3-h-old embryos), dechorionated, weighed, flash frozen in liquid nitrogen and stored at -80 °C, until approximately 200 mg has been collected. All subsequent steps are performed on ice or in a 4 °C cold room using prechilled buffers and pipettes.
Note: Alternative tissues, for example tissue culture cells transiently expressing GFP- or mCherry-expressing proteins, can be substituted.
1. Transfer the tissue to a Dounce homogenizer on ice.
2. Add 400 μl of an appropriate homogenising buffer (e.g., BRB80 + 0.1% IGEPAL + PMSF + Pi and PPi) and homogenize.
3. Centrifuge homogenate at 14,000 x g for 10 min in a microfuge at 4 °C.
4. Transfer supernatant to a Beckman tube and spin at 100,000 x g for 10 min at 4 °C.
5. Transfer supernatant to a fresh tube and spin again for 30 min at 100,000 x g.
Incubation of HSS with cl-AP
Transfer the HSS to a new 1.5 ml tube and incubate with 20 μl (1:1 slurry) of GFP-TRAP-Sulfo beads, overnight in cold room on rotating wheel.
Cleaving of Sulfo-beads
1. Wash beads with assay buffer (e.g., 1 ml BRB80 + 0.1% IGEPAL) 4 times (Step C2) to remove unbound and non-specifically bound proteins.
2. After removing the supernatant from the last wash, add 50 μl of assay buffer containing 50 mM DTT (final concentration), to cleave the disulfide bond in the spacer arm.
3. Incubate sample in orbital shaker at RT for 2 h.
4. Pellet the beads at 2,500 x g for 2 min in a microcentrifuge.
5. Transfer the supernatant (eluate) to a fresh tube using a Gel-Saver II pipette tip.

Note: The purified protein/protein complex can now be used directly for in vitro assays. If 50 mM DTT is found to interfere with a particular assay, it can be eliminated using a desalting column (Steps A1-A7).

Acknowledgments

This work was funded by University of Exeter (Proof of Concept), Lucy Green; University of Exeter (Carlota Palmer PhD studentship) Ammarah Tariq.

We thank Stephen Green and Mark Wood (Exeter, UK) for initial discussions about photocleavable and other tags, and Mary Munson (Massachusetts, US) for highlighting the potential of thiol-cleavable tags for native complex purification, as published by the Rout lab (Rockefeller, US) (Fridy et al., 2015). We thank Chromotek for the gift of GFP-binding protein; Jack Chen, Dan Hayward and Chris Sullivan for initial attempts to develop the Cl-AP technique.

The original research article where this protocol was used is Tariq et al., 2020.

Competing interests

No competing interests declared.

References

Fridy, P.C., Thompson, M.K., Ketaren, N.E. and Rout, M.P. (2015). Engineered high-affinity nanobodies recognizing staphylococcal Protein A and suitable for native isolation of protein complexes. Anal Biochem 15: 477:92-4.
Tariq, A., Green, L., Jeynes, J. C. G., Soeller, C. and Wakefield, J. G. (2020). In vitro reconstitution of branching microtubule nucleation. Elife 9: e49769.