| Popis: |
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. |
