[24] Nondenaturing polyacrylamide gradient gel electrophoresis

Publisher Summary Because the functional specificity of plasma lipoproteins resides in major part in the apolipoprotein moieties associated with the lipoproteins, techniques that combine size analysis, such as gradient gel electrophoresis, with apolipoprotein identification, either directly by immunoblotting or indirectly by immunoaffinity chromatography, appear highly promising for investigation of lipoprotein structure and function. This chapter discusses the application of gradient gel electrophoresis primarily to the analysis of HDL and LDL subpopulations and indicates approaches for more detailed identification of these subpopulations. Gradient gel electrophoresis entails the migration of charged particles through a matrix composed of increasing concentrations of polyacrylamide gel. The effective pore size of the matrix is progressively reduced as the gel concentration increases, resulting in differential retardation of the migrating charged particles. Although protein molecular weight determination is possible by gradient gel electrophoresis by use of appropriate calibration standards, such determination of lipoprotein molecular weights is still only approximate and requires validation by use of lipoprotein species with molecular weights determined by other methods.