Endonuclease G, Mitochondrial (ENDOG)?is a conserved nuclease encoded by nuclear DNA and localized in the mitochondrial intermembrane space. It plays dual roles in apoptosis and mitochondrial genome maintenance. During programmed cell death, ENDOG is released into the cytosol, cleaving nuclear and mitochondrial DNA at specific sites to facilitate apoptotic degradation. Structurally, it functions as a homodimer with Mg2?-dependent endonuclease activity. Beyond apoptosis, ENDOG participates in mitochondrial DNA replication, repair, and maternal genome processing during embryogenesis. Notably, ENDOG interacts with cytochrome?c?(CYCS) to synergistically amplify apoptosis. This complex enhances nucleolytic DNA degradation and modulates the mitochondrial permeability transition pore (MPTP), further promoting cell death.Thus a functional ELISA assay was conducted to detect the interaction of recombinant rat ENDOG and recombinant human CYCS. Briefly, ENDOG was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CYCS-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ENDOG pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant rat ENDOG and recombinant human CYCS was shown in Figure 1, the EC50?for this effect is 1.01ug/mL.