Extended Data Fig. 3: αCD8-PsT nanobody generation and recognition by αFLAG antibodies.
From: Single-cell analysis of regions of interest (SCARI) using a photosensitive tag
(a) Coomassie staining of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel showing the indicated nanobodies before and after labeling with the PsT, FITC or AF647. Band marked with the open triangle represents the PsT-labeled αCD8D−1 and band marked with the closed triangle represents a hydrolysis product, that has previously been described in C-terminal protein labeling39. Data obtained from a single experiment. (b) Binding of αFLAG antibodies to the FLAG-tag with or without the NPBF cage (αCD8D−1-PsT and αCD8D−1-T respectively). Cells labeled with αCD8D−1-PsT (left panel) or αCD8D−1-T (right panel) were stained with polyclonal αFLAG antibodies or αFLAG clone L5. Data obtained from a single experiment. (c) Specific binding of αFLAG antibody clone D6W5B to the uncaged FLAG-tag. Cells stained with αCD8D−1 nanobody conjugated to either the FLAG-tag without cage (αCD8D−1-T, upper panel) or the FLAG-tag containing the NPBF cage (αCD8D−1-PsT, bottom panel) were stained with αFLAG antibody clone D6W5B. Note that appreciable AF488 signal is only observed when the uncaged FLAG-tag is used. Representative data from 3 independent experiments are depicted.
