Protocol for the preparation of a segmental linear polyacrylamide gradient gel: simultaneous determination of Lp[a], LDL, and HDL particle sizes.

Abstract

We describe in this report a protocol for the preparation of a polyacrylamide gel system (S-GGE 2.8/8.30) that consists of two linear gradients designed for the simultaneous determination of the diameters of LDL and HDL from whole plasma. The lower gel consists of an 8-30% linear gradient which is optimum for the resolution of HDL subfractions and the upper gel consists of a 2-8% linear gradient to allow for the resolution of LDL and larger lipoprotein fractions such as Lp[a] and small VLDL. In contrast to other non-denaturing gradient gel systems which are based on protein staining, the present system uses lipid stain to specifically identify lipoproteins. This approach also allows the plasma to be pre-stained with immediate visualization of the lipid bands being possible at the completion of the electrophoretic run. Using commercially available gel casting equipment, the present gradient gel system can accommodate up to 21 lanes per gel. The inter-run and intra-run coefficients of variation for LDL particle size are 0.47 and 0.16%, respectively. The inter- and intra-run CVs for Lp[a] particle size are 0.92% and 0.89%, respectively. The inter-run and intra-run coefficients of variation for HDL2 and HDL3 particle size are 1.36% and 3.23%, respectively.