Human CLCA1 蛋白, His tag

CLCA1 (Calcium-activated chloride channel regulator 1) is a member of the calcium sensitive chloride conductance protein family. This protein is expressed as a precursor protein that is processed into two cell-surface-associated subunits. It has been reported that CLCA1 activates calcium-dependent chloride channel through the interaction with TMEM16A (anoctamin-1). Besides, there exits similarities between human and mouse TMEM16A in amino acid sequence with the identity of 89.66% . Thus, a functional ELISA assay was conducted to detect the association of recombinant human CLCA1 with recombinant mouse TMEM16A. Briefly, CLCA1 were diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to TMEM16A-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-CLCA1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37ºC. Finally, add 50 μl stop solution to the wells and read at 450nm immediately. The binding activity of CLCA1 with TMEM16A was in a dose dependent manner.