Extended Data Fig. 5: Characterization of nanobodies labeled with monomeric or dimeric PsT.
From: Single-cell analysis of regions of interest (SCARI) using a photosensitive tag
(a) SDS-PAGE analysis of nanobody-PsT generated under non-reducing or reducing conditions. Left panel: Coomassie stained SDS-PAGE gel showing the reduced (left) or non-reduced (right) versions of PsT-labeled αCD8D−1, αCD47D and αHLA-ID nanobodies. Right panel: AF594 fluorescence signal of the same gel. Note the increased size of PsT-nanobodies when coupled under non-reducing conditions as compared to PsT-nanobodies generated under reducing condition, indicating the presence of PsT dimers under non-reducing conditions (see also LC-MS and NMR spectra in Supplementary Information). Lower band in both conditions represents the non-fluorescent hydrolysis product that is formed during sortase reactions39. Data obtained from a single experiment. (b) Flow cytometric analysis of the uncaging of CD8+ T cells labeled with αCD8D−1-PsT nanobodies generated under reducing or non-reducing conditions (n = 2 technical replicates). Data were obtained from a single experiment.
