A Fast and Sensitive Method for Detection of Phospholipid-Binding Proteins on Nitrocellulose Membranes

Abstract

We describe a sensitive new method for specific detection and quantitation of phospholipid-binding proteins using two purified lipocortins as model proteins. The method consists of blotting proteins to nitrocellulose membranes followed by incubation with radiolabeled phospholipids and autoradiographic detection of bound phospholipids. It allows specific detection of phospholipid binding proteins to a threshold of about 100 ng/spot and their quantitation up to several micrograms as well as rapid analysis of specific phospholipid binding properties of individual proteins. If performed as a dot-blotting technique, the method permits the simultaneous analysis of a large number of samples and the establishment of Ca2+ and phospholipid binding curves. If blotting is preceded by electrophoresis, the method also allows specific detection and quantitative estimation of phospholipid binding proteins in crude biological preparations and may thus be useful for monitoring and analysis of these proteins in various biological samples.