Fig. 3
Detecting Antibody Specificity for NT and CT SIRT1 domains. (A) In-Vitro cleavage of SIRT1 was carried out by incubating 2µg flSIRT1 (110 kDa) with recombinant human Cathepsin B (rh. Cathepsin B), for 15 min at RT, to generate the 75SIRT1 NT-intact cleaved variant (ranging till 8-fold dilution) or loading 62–500 µg (pNT)/ 2000–250 µg (mCT) human full-length SIRT1. Each immunoblot was incubated with NT reactive (pNT) or CT-reactive (mCT) antibody. (B) The formulated ELISA method was designed to detect full-length SIRT1, using the sequence-specific antibodies mCT and pNT. (C) Sandwich ELISA was performed according to the set up in (B) with three antigens at a range of concentrations (100,50,25,12.5,6.25,3.125 ng/mL): (i) flSIRT1 (black circle), (ii) 75SIRT1 (pink circle), (iii) both flSIRT1 and 75SIRT1 at 50% v % (green rectangle). The right table exhibits the R2, p-value and standard curves for (i)-(iii), using Pearson’s correlation. Notably, R2 closer to 1 indicates a linear fit of the standard curve of the antigen to the antigen, which is seen with flSIRT1 (R2 = 0.9811), while 75SIRT1 R2 was 0.0936