Fig. 1

Antibody design and site-specific affinity binding: (A) The full-length SIRT1 protein (1-747aa) is illustrated with its N-terminal domain (blue rectangle), and C-terminal domain (brown rectangle). We designed peptides corresponding the NT a.a. SIRT1 sequence (composed of 2 peptides spanning ∼ 10-150aa) and the CT SIRT1 sequence (composed of 2 peptides spanning ∼ 530–747). To generate CT antibodies (mCT), CT peptides were injected to mice, while NT peptides were injected to rabbits to generated polyclonal NT-reactive antibody (pNT). (B) Right panel illustrates the sandwich ELISA method, wherein the NT fragments were detected with primary NT detection Ab (Millipore 07-131 or “a”; 1:2,000), following capture with mCT (n = 8). To detect CT (red ball) or NT (purple ball) peptides, we employed a sandwich ELISA, wherein mCT served as capture and Bethyl (CT -reactive; A300-688) served as a detection antibody. (C) Indirect ELISA was adapted from Batshon et al., 2020 [29]. Left scheme illustrates NT-peptide coated wells, incubated with pNT (1:1,000). Right panel indicates CT-peptide coated wells incubated with pNT (n = 6). (D) The net OD was detected for sandwich ELISA in panel B to assess the reactivity of mCT to the NT vs. CT peptide. Statistical significance determined by Unpaired T-test of net signal, two-tailed, P value = 0.0201. The net OD was detected for indirect ELISA in panel C to determine the specificity of pNT to NT or CT peptides. Statistical analysis was carried out using Mann-and-Whitney, assuming significance at p < 0.05