4.7.5. Serum Levels of p53 and Cytochrome c
The serum was centrifuged at 4 °C for 10 min at 1000× The Cytochrome c ELISA assay was performed by using a Cytochrome c Human ELISA Kit (Catalog No. ab119521, Abcam, Cambridge, UK). Briefly, 100 µL of serum samples were plated in duplicate in a 96-well microtiter plate pre-coated with anti-Cytochrome c monoclonal antibody. Then 50 µL of biotinylated detection antibody was added to each well. The plate was then incubated at room temperature for 2 h. After the washing step, 100 uL of streptavidin-horseradish peroxidase was added and incubated at room temperature for 1 h. The plate was then washed, followed by the addition of TMB substrate and stop solution. Optical density was measured by spectrophotometer at 450 nm (reference wavelength 620 nm) using a microplate reader.