S100 Calcium Binding Protein (S100) is a member of the S100 calcium-binding protein family, primarily expressed in cardiac and skeletal muscle cells. This 10.4 kDa protein plays crucial roles in calcium homeostasis, muscle contraction regulation, and energy metabolism through interactions with target proteins like titin and ryanodine receptors. S100A1's dysregulation is associated with cardiovascular diseases, including heart failure and hypertrophy. S100 directly binds ANXA6 in a calcium-dependent manner, modulating membrane repair processes.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human S100 and recombinant mouse ANXA6 .Briefly, biotin-linked S100 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to ANXA6-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µl stop solution to the wells and read at 450nm immediately. The binding activity of recombinant human S100 and recombinant mouse ANXA6 was shown in Figure 1, the EC50 for this effect is 0.09 µg/mL.