Calpain 2 (CAPN2) is a calcium-regulated, non-lysosomal thiolprotease that is known for its crucial role in catalyzing the limited proteolysis of substrates essential for cytoskeletal remodeling and signaling pathways. It belongs to the family of calpains, a group of enzymes that are activated by calcium ions.Besides,CAPN1 has been identified as an interactor of CAPN2, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human CAPN2 and recombinant rat CAPN1. Briefly, CAPN2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CAPN1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CAPN2 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 humanCAPN2 and recombinant rat CAPN1 was shown in Figure 1, the EC50?for this effect is 0.044ug/mL.