Figure 1. The binding activity of CKB with CKM.
Creatine Kinase, Brain (CKB) also known as CK-BB is a creatine kinase. CKB, consists of a homodimer of two identical brain-type CK-B subunits, is a cytoplasmic enzyme involved in cellular energy homeostasis, with certain fractions of the enzyme being bound to cell membranes, ATPases, and a variety of ATP-requiring enzymes in the cell. Besides, Creatine Kinase, Muscle (CKM) has been identified as an interactor of CKB, thus a binding ELISA assay was conducted to detect the interaction of recombinant human CKB and recombinant human CKM. Briefly, CKB were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to CKM-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CKB 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 CKB and CKM was shown in Figure 1, and this effect was in a dose dependent manner.