Journal of Lipid Research, Vol 31, 149-155, Copyright © 1990 by Lipid Research, Inc.
Analytical isoelectric focusing with immobilized pH gradients of human apolipoprotein E from very low density lipoproteins and total plasma
F Mailly, J Davignon and AC Nestruck
Hyperlipidemia and Atherosclerosis Research Group, Institut de Recherches, Cliniques de Montreal, Quebec, Canada.
A method for analytical isoelectric focusing (IEF) of apolipoprotein E
(apoE) in immobilized pH gradients (IPG) and immunodetection of the
separated isoforms has been developed for use with either very low density
lipoproteins (VLDL) or whole plasma. Both VLDL and plasma were sequentially
delipidated with 1,4-dioxane, acetone-ethanol, and ether. Neuraminidase
treatment preceded the delipidation when required. Using preformed plates,
pH 5.0-6.0 (LKB, Bromma) after rehydration with 6 M urea and dextran T-10,
the IPG focusing pattern of the common isoforms (E2, E3, E4) was found to
be equivalent to conventional IEF with the added resolution of the E4
disialo form. The use of self-poured narrower gradients permitted the
further resolution of the E4 monosialo form, a previously unrecognized
heterogeneity of the E2, E3, and E4 monosialo isoforms and differentiation
of the apoE2** mutant; all of these forms comigrate with the common
isoproteins in conventional IEF. Finally, the conditions for IPG of whole
plasma using apoE monoclonal antibodies and enzyme-conjugated anti-mouse
IgG for detection were established. Thus, IPG focusing is shown to be a
powerful method for resolution of the apoE sialoforms and apoE mutant
forms. The method has important implications in accurate and diagnostic
phenotyping. Moreover, it is a convenient method for phenotyping which
requires only very small volumes of plasma.
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