Opa1, also known as Mgm1 in yeast, is a mitochondrial member of the dynamin 
family. Unlike other dynamin family members, Opa1 has an N-terminal 
mitochondrial targeting sequence, suggesting that this protein is imported into 
mitochondria. Here, we describe biochemical techniques, such as mitochondrial 
isolation, digitonin extraction, a protease protection assay, and carbonate 
extraction, that were used to determine that mammalian Opa1 resides in the 
intermembrane space where it is tightly bound to the inner membrane. In 
addition, we describe bacterial expression of the Opa1 GTPase domain, methods 
for purification, and an in vitro assay for GTP hydrolysis.