Figure 4.

GFP and tdTomato nanobody performance in tissue IF and affinity purifications.A, GFP nanobodies were conjugated to Dynabeads and used to affinity purify Nup84-GFP and its associated NPC subcomplex from yeast. An anti-mCherry nanobody (LaM6) was used as a negative control for nonspecific background, and the LaG16-2 nanobody heterodimer used as the highest affinity nanobody reagent previously reported (14). Complexes were eluted in SDS and analyzed by Coomassie-stained SDS-PAGE. Band densities of Nup133, Nup84-GFP, Nup120, and Nup85/Nup145c were quantified and normalized to the LaG16-2 lane (n = 3). B, Briefly, 1 μg of GST-TEV was diluted in 0.2 to 5 ml of Escherichia coli lysate and isolated with glutathione- or nanobody-coupled Sepharose, with SDS elutions analyzed by Coomassie-stained SDS-PAGE. Recovery of the GST-tagged protein was quantified by densitometry (n = 3). C, new GFP nanobodies were labeled with Alexa Fluor 568 and used to stain brain sections of Thy1-GFPM mice. Quantitative comparisons of relative GFP and nanobody fluorescence are plotted at right. Fluorescence was normalized to maximum fluorescence in each channel. Plots were fitted with simple linear regression, and Pearson correlation coefficients (r) are indicated. The scale bars represent 100 μm. D, tdTomato nanobodies were labeled with Alexa Fluor 647 and used to stain sagittal brain sections of ChAT-Cre::Ai14 mice, with tdTomato expressed in cholinergic neurons. Quantification is shown as in (C). The scale bars represent 100 μm. E, a GFP nanobody heterodimer was labeled with Alexa Fluor 647 and used for Thy1-GFPM brain section staining as in (C). The scale bars represent 100 μm. IF, immunofluorescence; NPC, nuclear pore complex; TEV, tobacco etch virus.