Figure 6. Immunofluorescence with site-specifically labeled anti-Nup nanobodies.
(a) Xenopus XL177 cells were digitonin-permeabilized and stained with anti-Nup nanobodies carrying a single N-terminal Alexa Fluor 647 maleimide dye before fixation and DAPI staining. A characteristic nuclear rim stain indicates labeling of NPCs. A nanobody raised against Escherichia coli Maltose-binding protein (MBP) served as a negative control. (b) Labeling of the anti-GFP nanobody Enhancer with Alexa Fluor 647 NHS ester at lysines or at three engineered cysteines using Alexa Fluor 647 maleimide. Labeling introduces a size shift in SDS–PAGE. Detection was either by Coomassie staining or by in-gel fluorescence. (c) Staining of HeLa cells stably expressing GFP-tagged Nup153 with the anti-GFP nanobody labeled via NHS ester or maleimide Alexa Fluor 647. The nanobody TP377, raised against Xenopus (x)Nup98, does not cross-react with human Nup98 and served as a negative control. The NHS-labeled GFP nanobody produced strong background-staining, while its maleimide-labeled version yielded bright nuclear rim stains. (d) Staining of XL177 cells with nanobodies labeled with Alexa Fluor 647 either at their internal lysine residues (NHS ester dye) or via engineered cysteines (maleimide dye). Note that the widely used anti-GFP nanobody Enhancer produces significant background staining when labeled via lysines but not when using engineered cysteines and a maleimide dye. All nanobodies were used at a concentration of 10 nM and all images were obtained under identical settings. DOL, degree of labeling.