Active Mitofusin 2 (MFN2)

Mitofusin 2 (MFN2) is a dynamin-like GTPase protein primarily located on the outer membrane of mitochondria. It is a master regulator of mitochondrial dynamics, mediating the fusion of mitochondria to maintain their shape, distribution, and functional health. This process is crucial for cellular energy metabolism, calcium signaling, and overall stress response. MFN2 is also uniquely present on the endoplasmic reticulum (ER), where it plays a pivotal role in forming physical tethers between the ER and mitochondria, establishing specialized domains known as Mitochondria-Associated Membranes (MAMs). These contact sites are essential for lipid transfer, apoptotic signaling, and coordinated calcium flux. MFN2 directly binds to VDAC1 on the mitochondrial surface to bridge the ER-mitochondrial interface, facilitating inter-organelle communication.To detect the activity of recombinant MFN2, a functional ELISA assay was performed to evaluate the interaction between recombinant rat MFN2 and recombinant human VDAC1.Briefly, biotin-linked MFN2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to VDAC1-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 50µl stop solution to the wells and read at 450nm immediately. The binding activity of MFN2 and VDAC1 was shown in Figure 1, the EC50 for this effect is 0.12µg/mL.