Fig. 1: Proximity-dependent biotinylation using Nfasc antibodies. | Nature Communications

Fig. 1: Proximity-dependent biotinylation using Nfasc antibodies.

From: Antibody-directed extracellular proximity biotinylation reveals that Contactin-1 regulates axo-axonic innervation of axon initial segments

Fig. 1

a Illustration of the antibody-directed proximity biotinylation strategy. Anti-Nfasc antibodies bind to Nfasc, while HRP-conjugated secondary antibodies bind to the Nfasc antibodies. Addition of biotin phenol (biotin tyramide) in an H2O2 containing diluent results HRP-mediated conversion of the biotin phenol to an active radical biotin phenoxyl that covalently adds the tyramide biotin to extracellular tyrosine residues. Omission of the primary anti-Nfasc serves as a control. After stringent solubilization and affinity capture by streptavidin-conjugated magnetic beads. Biotinylated proteins are then identified by mass spectrometry. Created with Biorender.com. b Fluorescence imaging of DIV14 rat hippocampal neurons labeled by Nfasc-BAR or a control condition (no primary Ab). Nfasc fluorescence (green) enrichment defines the AIS. Hoechst (blue) labels nuclei, and biotinylated proteins are detected using Alexa594-conjugated streptavidin (red). N = 3 independent experiments. Scale bars, 20 μm.

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