Active Calreticulin (CALR)

Calreticulin (CALR) is an endoplasmic reticulum (ER)-resident protein involved in a spectrum of cellular processes. In healthy cells, CALR operates as a chaperone and Ca2  buffer to assist correct protein folding within the ER. Calreticulin is also found in the nucleus, suggesting that it may have a role in transcription regulation. Systemic lupus erythematosus is associated with increased autoantibody titers against calreticulin. Recurrent mutations in calreticulin have been linked to various neoplasms, including the myeloproliferative type. Adhesive glycoprotein THBS1 has been shown to play roles in platelet aggregation, angiogenesis and tumorigenesis, the protein is an interactor of CALR, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human CALR and recombinant rat THBS1. Briefly, biotin-linked CALR were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to THBS1-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 CALR andTHBS1 was shown in Figure 1, the EC50 for this effect is 0.018 ug/mL.