Binding of serpins to immobilized phospholipids and phospholipids in suspension

Abstract

Binding of serine protease inhibitors (serpins) to nonprotein ligands such as glycosaminoglycans or phospholipids has been shown to modify their inhibitory activity and—at least in the case of SERPINA5—to mediate serpin internalization into cells. Also phospholipid functions may be altered when bound to serpins or other proteins. By interacting with phospholipids, serpins might influence a variety of cellular functions. Binding of proteins to phospholipids can be studied by several methods. Here we describe solid-phase assays, in which pure phospholipids are immobilized on nitrocellulose membranes, PVDF membranes, or microtiter plates. Bound proteins are detected with specific antibodies and labeled secondary antibodies. We also describe a method visualizing binding of phospholipids in suspension by non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by Western blotting.