Western Blotting of Membrane-Bound Proteins in Yarrowia lipolytica.

Abstract

With the discovery of Western blotting as first described by Towbin et al. in 1979, the transfer and visualization of electrophoretically separated proteins on membranes has become the de facto method for the qualitative and quantitative detection of proteins of interest. In this method, proteins are resolved by electrophoresis on a polyacrylamide gel, followed by a transfer of the separated proteins onto a nitrocellulose or polyvinyl difluoride (PVDF) membrane. Once immobilized on these membranes, the protein of interest can be detected and visualized by exploiting antigen-antibody interactions. However, not all proteins are amenable to easy detection by Western blotting. Integral membrane proteins are a class of proteins that are attached to a biological membrane through a series of transmembrane segments that span the width of the membrane. Due to the inherent hydrophobicity of these proteins and their tendency to aggregate, the characterization and detection of these proteins can be challenging. In this methods chapter, we present a protocol for the easy detection and quantification of these proteins in the industrially important oleaginous yeast Yarrowia lipolytica. The first protocol describes a Western blotting procedure to quantify soluble cytosolic proteins of interest in Yarrowia lipolytica from its total cell lysate. The second protocol describes modifications to the first that are done to enhance detection and quantification of membrane-bound proteins in Yarrowia lipolytica from its total cell lysate, without the need for isolating the membrane-bound proteins, for use in Western blotting. The immunoblotting strategies described here should serve as an efficient and simple guide to quantify both cytosolic and the intractable membrane-bound proteins in Yarrowia lipolytica.