Orthogonal staining of Nanobody immune libraries displayed on yeast

Yeast cells displaying a Nanobody by use of our optimised system (Fig. 2) were inoculated in SDCAA medium (20 g glucose, 6.7 g yeast nitrogen base, 5 g bacto casamino acids, 5.4 g Na₂HPO₄ and 8.56 g NaH₂PO_4•H₂O _dissolved in 1 litre of H₂O, supplemented with 10 ml of Gibco^TM Penicinillin-Stereptomycin, 10,000 U/ml Thermo Fisher Scientific) and grown to an OD₆₀₀ of 1. When displaying libraries, the inoculum contained at least ten times more cells than the library size. For induction, cells were harvested at 4,000 g and resuspended in SGCAA medium (20 g galactose, 6.7 g yeast nitrogen base, 5 g bacto casamino acids, 5.4 g Na₂HPO₄ and 8.56 g NaH₂PO_4•H₂O in 1 litre of H₂O, supplemented with 10 ml of Gibco^TM Penicinillin-Stereptomycin, 10,000 U/ml Thermo Fisher Scientific) and grown at 30 °C overnight at 120 rpm. For orthogonal staining, a total of 4 × 10⁷ induced yeast cells were washed twice in cold PBS supplemented with 0.2% (w/v) BSA at pH 7.4 (PBS–BSA), harvested at 4,000 g and stored on ice. For the covalent labelling of ACP, cells were routinely resuspended in 100 µl of reaction buffer (50 mM HEPES, pH 7.4, 10 mM MgCl₂) supplemented with 1 µM of the Sfp synthase and 1 µM of the CoA derivative of choice: CoA-547 fluorophore, CoA-647 fluorophore, CoA-biotin (New England BioLabs) or synthesised CoA-Alexa488. After 60 min of staining in a rotating 50 rpm at room temperature, the labelled cells are washed three times with 500 µl of ice-cold PBS–BSA and can be stored for days on ice.