Simplified isoelectric focusing/immunoblotting determination of apoprotein E phenotype.
We developed a rapid, accurate method for phenotyping apoprotein E that can be used for large-scale population studies. In this method, adapted from the method of Kamboh et al. (J Lipid Res 1988;29:1535-43), 10-microL plasma samples are incubated with dithiothreitol and Tween-20 for 15 min and then applied to 5% polyacrylamide gels containing ampholyte (pH 4.5-8) and urea (3 mol/L). After 2 h of isoelectric focusing, the apoprotein E bands are made visible by immunoblotting. Utilizing whole plasma, this method does not require time-consuming ultracentrifugation, delipidation of samples, or dialysis. Small amounts of plasma are required, electrofocusing time is short, and as many as 160 samples can be processed per day. Identification of phenotype is easily accomplished by noting the location and number of protein bands instead of their intensity. Because identification of phenotype is not affected by sialylation, neuraminidase treatment is not necessary. Agreement in identification of 301 individuals from blinded duplicates was 96%, and there was 98% concordance of results for 431 samples that had undergone genetic typing. This method is thus well suited for large-scale population studies.
Kataoka
S
S
Medlantic Research Institute, Washington, DC 20010-2933.
Paidi
M
M
Howard
B V
BV
D001057:Apolipoproteins E / Q000032:analysis*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D010641:Phenotype
D001057:Apolipoproteins E