Gradient gel electrophoresis-immunoblot analysis (GGEI): a sensitive method for apolipoprotein profile determinations.
A method is described which will determine the distribution of individual apolipoproteins within the HDL subclasses. This method requires 1-2 microliters of plasma per determination and involves six steps: 1) electrophoresis of samples on non-denaturing 2-30% concave acrylamide gradient gels; 2) electrophoretic transfer of the lipoproteins to charge-modified nylon membranes; 3) fixation of the transferred lipoproteins with glutaraldehyde; 4) immunolocalization of the apolipoproteins with iodinated monospecific antibodies; 5) autoradiography followed by densitometry; and 6) reduction of the data to provide a plot of percent distribution versus particle size. When this method was applied to the analysis of rat apolipoproteins, differences were noted in the distribution of apoA-I, apoA-IV, and apoE. The majority of apoA-I was localized to HDL particles between 9 and 12 nm in diameter, with a median diameter of 10.0 nm, while apoE resided on substantially larger particles with a median diameter of 12.5 nm. ApoA-IV could be localized to three distinct areas: an HDL particle with a median diameter approximately 0.4 nm larger than apoA-I HDL, a particle smaller than albumin (lipoprotein-free apoA-IV), and a particle of 7.6 nm that does not appear to contain apoA-I or apoE.
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D001053:Apolipoproteins / Q000097:blood*; D001345:Autoradiography; D003720:Densitometry; D004586:Electrophoresis / Q000379:methods; D005976:Glutaral; D006801:Humans; D007163:Immunosorbent Techniques; D008297:Male; D051381:Rats
D000911:Antibodies, Monoclonal; D001053:Apolipoproteins; D005976:Glutaral