Abstract
Conventional determination of apolipoprotein E isomorphs comprises ultracentrifugation of 1-5 ml serum, delipidation of very low density lipoproteins (VLDL), and isoelectric focusing (IEF) in polyacrylamide gels. In order to reduce the sample volume and to avoid nonspecific protein bands, immunoblotting was proposed. Now we describe a methodological variant that uses 25 microliters serum, replaces ultracentrifugation by precipitation of apoE-containing lipoproteins with polyethylene glycol, and delipidation by dissolution in detergent. IEF is carried out in agarose. This allows specific immunofixation of apoE-containing bands with 10 microliters antiserum per sample. This method yields apoE patterns that are specific and well resolved. Also, it offers considerable savings of time and equipment involved.
Highlights
We have developed a methodological variant that simplifies several steps of the conventional procedure: I) ultracentrifugation is replaced by precipitation of apoEcontaining lipoproteins, 2) delipidation is replaced by dissolution of the precipitate by means of a detergent, 3)isoelectric focusing (IEF) in polyacrylamide gels is replaced by IEF in agarose, which 4) allows specific immunofixation with antiserum against apoE
Two hundred ml of 2.5% agarose containing 11% sorbitol is prepared in a wide Erlenmeyer flask by gentle boiling for 1 h
Using a prewarmed 20-ml pipette, the agarose is poured between the warm glass plates which are held at an angle of about 45O
Summary
Zentrallabor der Klinik der Albert-Ludwigs- Unibersitat,* Freiburg, Germany and Abteilung Sport- und Leistungsmeditin,t Freiburg, Germany. We have developed a methodological variant that simplifies several steps of the conventional procedure: I) ultracentrifugation is replaced by precipitation of apoEcontaining lipoproteins, 2) delipidation is replaced by dissolution of the precipitate by means of a detergent, 3)IEF in polyacrylamide gels is replaced by IEF in agarose, which 4) allows specific immunofixation with antiserum against apoE. This procedure is relatively easy, quick, very specific and requires 25 pl of serum and 10 pl of antiserum per sample
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