Nanobody Conjugation and Affinity Isolation

Recombinant nanobodies were conjugated to epoxy-activated magnetic Dynabeads (Life Technologies), with minor modifications to published IgG coupling conditions^{57 (link)}. 10 μg recombinant protein was used per 1 mg of Dynabeads, with conjugations carried out in 0.1 M sodium phosphate, pH 8.0 and 1 M ammonium sulfate, with an 18–20 hour incubation at 30°C. Affinity isolations of yeast Nup84-GFP were carried out as previously described, using binding buffer consisting of 20 mM HEPES, pH 7.4, 500 mM NaCl, 2 mM MgCl₂, 0.1% CHAPS, 0.1 M PMSF, and 3 μg/ml pepstatin A^{57 (link)}. For each experiment, 50 μl of bead slurry was used with 0.5 g of yeast cells. Similar conditions were used for HTB2-mCherry isolations (from yeast with HTB2 genomically tagged at the C-terminus with mCherry^{58 (link)}), except lysate was sonicated 4 times for 10 s before centrifugation, and the binding buffer consisted of 20 mM HEPES, pH 8.0, 300 mM NaCl, 110 mM KOAc, 0.1% Tween-20, 0.1% Triton X-100, 0.1 M PMSF, and 3 μg/ml pepstatin A. Isolations of RBM7-GFP from HeLa cells were performed as previously described^{4 (link)}. 10 μl of bead slurry was used with 100 mg of cells, using a binding buffer of 20 mM HEPES, pH 7.4, 300 mM NaCl, 0.5% Triton X-100, with cOmplete Protease Inhibitor, EDTA-free (Roche).
To determine affinity isolation yields, samples of resuspended lysate were taken before and after Dynabead binding. These were run on a 4–12% Novex Bis-Tris gel in MES running buffer (Life Technologies), and probed by Western blotting using mouse anti-GFP antibody (Roche, cat. no. 11 814 460 001) diluted 1:1,000 in TBST/2% dry milk and an anti-mouse, HRP-conjugated secondary (GE Healthcare, cat. no. NA931V) diluted 1:3,000 in TBST/2% dry milk. Signals were quantified using ImageJ software.