Activating Transcription Factor 6 (ATF6) is a key protein in the endoplasmic reticulum (ER) stress response. It is a transmembrane protein located in the ER membrane. Under normal conditions, ATF6 is bound to Heat Shock 70kDa Protein 5 (HSPA5), also known as GRP78, and remains inactive.When ER stress occurs due to the accumulation of unfolded or misfolded proteins in the ER lumen, HSPA5 preferentially binds to these abnormal proteins, releasing ATF6. The released ATF6 then translocates to the Golgi apparatus, where it is cleaved by proteases. The cleaved ATF6 fragment enters the nucleus and activates the transcription of genes involved in the unfolded protein response, helping the cell to restore ER homeostasis.Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse ATF6 and recombinant mouse HSPA5. Briefly, ATF6 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to HSPA5-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ATF6 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 mouse ATF6 and recombinant mouse HSPA5 was shown in Figure 1, the EC50?for this effect is 0.031ug/mL.