Stromal Interaction Molecule 1 (STIM1) is a single-pass transmembrane protein located primarily in the endoplasmic reticulum (ER) membrane. It functions as a critical calcium sensor in the process of store-operated calcium entry (SOCE). When ER calcium stores are depleted, STIM1 undergoes conformational changes, oligomerizes, and translocates to ER-plasma membrane junctions where it interacts with and activates Orai channels, facilitating extracellular Ca2? influx. This process is essential for various cellular functions including immune response, muscle contraction, and gene expression. STIM1 directly binds to the Inositol-1,4,5-Trisphosphate Receptor Type 1 (ITPR1), an ER-resident calcium release channel, to regulate its stability and calcium release activity.
To detect the activity of recombinant STIM1 , a functional ELISA assay was performed to evaluate the interaction between recombinant human STIM1 and recombinant human ITPR1.Briefly, STIM1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ITPR1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-STIM1 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 STIM1 and recombinant human ITPR1 was shown in Figure 1, the EC50?for this effect is 0.08 μg/mL.