Band 3 (BND3), also known as Anion Exchanger 1 and SLC4A1, is a 90-100 kDa polytropic membrane protein that ?is part of the anion exchanger (AE) family and is expressed in the erythrocyte plasma membrane, where it mediates the exchange of the cellular HCO3- with CI- in plasma. BND3 is required for normal flexibility and stability of the erythrocyte membrane and for normal erythrocyte shape via the interactions of its cytoplasmic domain with cytoskeletal proteins, glycolytic enzymes, and hemoglobin. Scaffold protein Ankyrin 3, Node Of Ranvier (ANK3) can Interacts with BND3, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat BND3 and recombinant human ANK3. Briefly, biotin-linked BND3 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to ANK3-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 50 μl stop solution to the wells and read at 450 nm immediately. The binding activity of recombinant rat BND3 and recombinant human ANK3 was shown in Figure 1, the EC50?for this effect is 0.35 ug/mL.