Epitope mapping of syndecan-4 antibody (sc-12766) and design of a specific blocking peptide.

<p>A) The antibody epitope was identified by overlaying an array of immobilized overlapping 20-mer human syndecan-4 peptides with anti-syndecan-4 (upper panel). Amino acids relevant for anti-syndecan-4 recognition are given. Underlined amino acids indicate the core epitope. Immunoblotting without anti-syndecan-4 was used as control (lower panel). B) Alignment of human vs. bovine and mouse syndecan-4 protein sequences. Black boxes indicate identical amino acids (DNA Star, Madison, Wisconsin). C) Amino acid sequences relevant for anti-syndecan-4 binding were synthesized on a membrane and overlaid with anti-syndecan-4 pre-incubated without (left panel) or with the blocking peptide (middle panel). Anti-syndecan-4 was omitted in the negative control (right panel). D) Staining of myoblasts and myotubes after three days in differentiating medium. Cells were fixed with ice-cold ethanol and immunostained with anti-syndecan 4 (sc-12766) alone (diluted in Pierce Immunostain Enhancer for increased antibody binding), or in combination with various concentrations of blocking peptide (2-330-660 μM), followed by Alexa 546-conjugated goat anti-mouse (red) before fluorescence microscopy analysis (ZEISS Axio Observer Z1 microscope). Scale bar: 50 μm. E) Control experiment with secondary antibody (SA) alone show little unspecific binding. Cells stained with anti-syndecan-4 followed by Alexa 546-conjugated goat anti-mouse (upper panel), or with secondary antibody (Alexa 546-conjugated goat anti-mouse) alone (lower panel) before fluorescence microscopy analysis (ZEISS Axio Observer Z1 microscope). Nuclei were stained with DAPI (blue). All images were captured using the same settings.