Apolipoprotein E Phenotyping from Plasma by Isoelectric Focusing and Immunoblotting

Abstract

ETO, M., WATANABE, K., MORIYAMA, T. and MAKINO, I. Apolipoprotein E Phenotyping from Plasma by Isoelectric Focusing and Immunoblotting. Tohoku J. Exp. Med., 1990, 160 (4), 301-309-A method for apolipoprotein (apo) E phenotyping directly from plasma by isoelectric focusing (IEF) and immunoblotting was confirmed. Ten μl plasma were delipidated. IEF in 5% polyacrylamide flat gel with 6.4mol/l urea and 2.8% pharmalyte (PH 4-6.5) was carried out at 3, 000V for 1hr. Seventeen samples were applied per one flat gel, and IEF of two flat gels was made. Then, Western blotting on nitrocellulose membrane was done at 75V for 3hr. Immunostaining was performed using goat-anti-human apo E as first antibody and biotinylated anti-goat IgG as second antibody, and 4-chlorodel-1-naphthol as a substrate. In approximately 5% of the samples, we had difficulty in discriminating between homozygotes and heterozygotes (i.e., apo E3/3 and apo E3/2, or apo E4/4 and apo E4/3) because of equally strong sialated band, but this problem was solved by sialidase treatment of plasma before delipidation. As a result, six apo E phenotypes were clearly demonstrated. Apo E phenotyping of 34 samples could be made simultaneously in 2 days. It is concluded that the polyacrylamide gel IEF and immunoblotting method is useful for apo E phenotyping if it is made up for by sialidase treatment.