Abstract
A rapid micromethod for apo E phenotyping directly from plasma has been established. Ten μl plasma were delipidated, dried and then solubilized in 10 mM Tris-HCl buffer with 8 M urea. Isoelectric focusing (IEF) in 5% polyacrylamide flat gel with 6.4M urea and 2.8% pharmalyte (PH 4-6.5, Pharmacia) was carried out, as reported previously. After IEF, the flat gel was washed in a blot buffer (0.7% acetate), followed by Western blotting on nitrocellulose membrane in the same blot buffer using the Trans-Blot Cell (Bio Rad Co.). Then, immunostaining was performed using goat-anti-human apo E (Daiichi Chemicals) as first antibody and biotinylated anti-goat lgG as second antibody, VECTASTAIN ABC Kit (VECTOR Laboratories), and 4-chloro-1-naphthol as a substrate. As a result, six apo E phenotypes were clearly demonstrated. It took only 2 days for apo E phenotyping. Thus, this method is suitable for routine examination and large scale screening for studying the relationship between apo E phenotypes and hyperlipoproteinemia and atherosclerosis
