Native–Native 2D Gel Electrophoresis for HDL Subpopulation Analysis

Abstract

High-density lipoproteins (HDLs) are a heterogeneous mixture of lipoprotein particles with densities ranging from 1.063 to 1.021 g/ml. The various lipoprotein particles present in HDL have not yet been completely characterized, largely due to methodological difficulties. Asztalos and Schaefer have developed a powerful native–native 2D gel method to analyze HDL subpopulations with high resolution. In this technique, native HDL particles present in plasma are separated electrophoretically by charge in the first dimension and then by size in the second dimension. The native particles in the gel are then transferred onto a membrane using traditional Western blotting techniques. After probing the membrane with, for example, apoA-I antibodies, subpopulations of HDL particles containing apoA-I can be visualized. Traditionally, midi-sized native gels are poured manually in the laboratory and running, blotting, and probing the gels is a tricky and laborious procedure that involves the use of ¹²⁵I-labeled antibodies. Here we present a streamlined native–native 2D gel electrophoresis and blotting method using minigels. Traditional antibody incubation and chemiluminescent methods can be used for detection and use of ¹²⁵I is not required. This update of the Asztalos and Schaefer native–native 2D gel protocol renders the procedure more accessible to the nonspecialist.