4.5. Measurement of SIRT1
SIRT1 quantification was performed using the double sandwich ELISA technique. We used a monoclonal anti-human SIRT1 antibody and a biotinylated detection antibody. Samples and biotinylated antibodies were added to ELISA plate wells and then washed with buffer. Then, avidin–peroxidase conjugate was added to the wells, and the corresponding substrate was added. The reaction mixture turned blue and then turned yellow upon the addition of acid. The absorbance of the colored compound was read at 450 nm on a Multiskan Go Microplate spectrophotometer (Thermo Scientific, Denver, CO, USA).