Following primary antibodies were used: rabbit anti-brevican “neo” (Rb399) (custom-made; rat neo-epitope QEAVESE; see Valenzuela et al., 2014 (link); 1:500), polyclonal sheep anti-human/rat brevican (AF4009, R&D Systems, Minneapolis, United States, 1:1000), polyclonal sheep anti-rat/mouse neurocan (AF5800, R&D Systems, Minneapolis, United States, 1:1000), and the antibodies from the ELISA kits (see above).
To prove if the neo-antibody, which was originally produced against the neo-epitope of the rat N-terminal brevican fragment, is also detecting the human brevican fragment neo-epitope EATESE we performed a competition assay with pre-incubation of the primary antibody with a 100-fold molar excess of the peptide Biotin-Ahx-CGQEAVESE containing the immunogen for 1 h at RT. As shown inSupplementary Figure 1 , the 60 kDa band detected by this antibody in human CSF samples is clearly diminished in the peptide competition condition, demonstrating its specificity.
As secondary antibodies we used peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H + L) and peroxidase-conjugated AffiniPure donkey anti-sheep IgG (H + L) (Jackson ImmunoResearch, Cambridgeshire, United Kingdom, 1:2000).
To prove if the neo-antibody, which was originally produced against the neo-epitope of the rat N-terminal brevican fragment, is also detecting the human brevican fragment neo-epitope EATESE we performed a competition assay with pre-incubation of the primary antibody with a 100-fold molar excess of the peptide Biotin-Ahx-CGQEAVESE containing the immunogen for 1 h at RT. As shown in
As secondary antibodies we used peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H + L) and peroxidase-conjugated AffiniPure donkey anti-sheep IgG (H + L) (Jackson ImmunoResearch, Cambridgeshire, United Kingdom, 1:2000).
Free full text: Click here
