Analysis and quantitation of biotinylated apoB-containing lipoproteins with streptavidin-Cy3

Non denaturing gradient gel electrophoresis (GGE) is commonly used to analyze the size distribution of lipoprotein particles. Its relatively low sensitivity and linear dynamic range limit use of GGE to quantify protein content of lipoproteins. We demonstrate a new high sensitivity method for analysis and quantitation of biotinylated apolipoprotein B (apoB)-containing lipoproteins using a fluorescent streptavidin-Cy3 conjugate and non covalent preelectrophoretic binding. Forty-four lipoprotein subfractions spanning the VLDL and LDL particle spectrum subfractions (11 each from four human subjects) were prepared by density gradient ultracentrifugation. An aliquot of each sample was biotinylated and GGE was performed. Gels also were stained for lipid with Oil Red O (32 samples) and for protein with Coomassie Brilliant Blue (30 samples). There was a significant relationship between the Cy3 fluorescent label area under the curve and the mass of apoB (P < 0.02-0.004) and total cholesterol (P < 0.03-0.004). Particle diameters of each absorbence/fluorescent peak were comparable between Oil-Red O and streptavidin-Cy3 treated biotinylated lipoproteins (+/-3.54 A, P = 0.3). Biotinylation and prestaining of lipoprotein particle with streptavidin-Cy3 provides a new fluorescence-based method for detection and quantitative analysis of lipoprotein subspecies by gradient gel electrophoresis.