Minichromosome Maintenance Complex Component 5 (MCM5) is an essential eukaryotic protein and a critical component of the hexameric MCM complex (MCM2-7). This complex acts as the core replicative helicase, responsible for unwinding double-stranded DNA at replication forks during the S phase of the cell cycle. Similar to other MCM proteins, MCM5 contains conserved ATPase motifs that supply the mechanical force required for DNA unwinding. Its expression is tightly regulated throughout the cell cycle, with levels peaking during the G1/S phase transition. Notably, MCM5 has emerged as a valuable biomarker in cancer diagnostics: its aberrant expression and detection in bodily fluids such as urine often signal dysregulated cell proliferation and malignant transformation.Besides,Fas Associating Death Domain Containing Protein (FADD) has been identified as an interactor of MCM5, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human MCM5 and recombinant human FADD. Briefly, MCM5 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FADD-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-MCM5 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 human MCM5 and recombinant human FADD was shown in Figure 1, the EC50?for this effect is 0.30 μg/mL..