Native Agarose Gel Electrophoresis of Multiprotein Complexes

INTRODUCTION

An important tool for the biochemist is the ability to analyze proteins in their native state. Electrophoresis of proteins and protein–protein complexes in native agarose gels using a horizontal gel apparatus is described here. The procedure is simple to set up, takes a short time to run, and avoids the use of toxic components. The gel is run in a submerged horizontal platform, with the wells positioned in the center of the gel. Proteins with a pI lower than the buffer pH carry a net negative charge and migrate toward the anode, whereas proteins with a pI higher than the buffer pH carry a positive charge and migrate toward the cathode. Proteins with different molecular weights and pI values can be tested, as can proteins that form complexes, whether they be two pure proteins forming a complex or a complex formed after incubating a pure protein with a crude extract. Once a complex is formed, it can be cut from the gel and the components can be isolated. This method does not replace isoelectric focusing (IEF) gels because it does not determine the pI of a protein, but it does facilitate the detection of protein–protein complexes and may give information on the protein’s charge at a defined pH.