Figure. The binding activity of CAPN3 with TTN.
Calpain 3 is a calcium-dependent cysteine protease mainly expressed in skeletal muscle. In humans, calpain 3 is encoded by the CAPN3 gene. This gene encodes a muscle-specific member of the calpain large subunit family that specifically binds to titin. Mutations in this gene are associated with limb-girdle muscular dystrophies type 2A. Alternate promoters and alternative splicing result in multiple transcript variants encoding different isoforms and some variants are ubiquitously expressed. Besides, Titin (TTN) has been identified as an interactor of CAPN3, thus a binding ELISA assay was conducted to detect the interaction of recombinant human CAPN3 and recombinant human TTN. Briefly, CAPN3 were diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to TTN-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CAPN3 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 37℃. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of CAPN3 and TTN was shown in Figure 1, and this effect was in a dose dependent manner.