Optimal epitope mapping for peptides 42 and 112 using sequentially truncated peptides identified the respective 9-mer binding epitopes as LVGPTPVNI and YYDPSKDLI (Fig. 2E).
To confirm the binding epitope as mapped by the peptide array experiment, a GST-fusion construct containing SKIP residues 43 79 was employed for GST pull-down experiments using purified PPIL1 protein.
Due to the overlapping epitopes of Fab AbD1556 and BMP-2 on BMPR-IA one can think of the Fab AbD1556 binding epitope as similar to an anti-idiotypic molecule mimicking BMP-2.
However, due to limited understanding of the molecular structure of antibody binding sites and antigenic epitopes, as well as the intermolecular binding forces that come into play, the traditional 'trial and error' method used to develop antibodies still remains the method of choice.
The traditional view of protein ligand binding treats a protein as comprising distinct binding epitopes on the surface of a degenerate structural scaffold, largely ignoring the impact of a protein's energy landscape.
Firstly, receptors are internalized upon MHC class I ligand-binding in trans, and secondly, Ly49 - MHC cis interactions masks the antibody binding epitopes on the Ly49 receptors, which appears as a lower detected expression level [19], [21], [22], [23].
Such competition assays are complicated by steric hindrance between Abs binding physically adjacent epitopes as well as more subtle positive and negative conformational effects that occur upon Ig binding to HA (Lubeck and Gerhard, 1982).
To more clearly define the binding epitope, the Lc domain of BoNT/A as well as smaller fragments were cloned into E. coli and expressed as GST fusion proteins (Figure 2).
However, given that multiple linear and often short, cryptic motifs and conformational epitopes, as well as binding partners and post-translational modifications, can finely tune subcellular sorting, experimental evidence is expected to shed light on the SCL, interactions and role in trafficking of this novel protein family.
We defined minimum binding epitopes for commercial antibodies recognizing the modified and unmodified H2B peptides.
