Active Fibronectin (FN)

Fibronectin (FN) is a high-molecular weight (~440kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Fibronectins bind cell surfaces and various compounds including collagen, fibrin, heparin, DNA, and actin. Fibronectin has numerous functions. For example, it involved in cell adhesion, cell motility, opsonization, wound healing, maintenance of cell shape, and so on. Besides, Decorin (DCN) has been identified as an interactor of FN, thus a binding ELISA assay was conducted to detect the interaction of recombinant human FN and recombinant human DCN. Briefly, FN were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to DCN-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-FN 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 of FN and DCN was shown in Figure 1, and this effect was in a dose dependent manner.

As FN has the function of cell adhesion, we measure the activity of FN by the ability of the immobilized protein to support the adhesion of NIH-3T3 mouse embryonic fibroblast cells. When 5 x 104 cells/well are added to recombinat human FN coated plates (10 µg/mL with 100 µL/well), approximately 70-80% will adhere after 3 hour incubation at 37℃. The adhesion of NIH-3T3 after 3 hour incubation at 37℃ observed by inverted microscope was shown in Figure 2.