Sodium dodecyl sulphate gel electrophoretic preparation of protein standard human apolipoprotein B-48

Abstract

Quantitation of plasma apo B-48 is currently performed by densitometric analysis of SDS–PAGE zones stained with Coomassie Brilliant Blue, using standard solutions of purified apo B-48. Here, preparative gel electrophoresis with a continuous elution system was used for purifying apo B-48. A chylomicron fraction was isolated by 107 000 g ultracentrifugation of a chylous ascite. The proteins were delipidated and precipitated in ethanol–diethyl ether (3:1, v/v), subjected to preparative electrophoresis in a 5% polyacrylamide gel and eluted in 0.1% SDS. The peak containing apo B-48 was eluted at a retention time of 445–480 min. The purity of apo B-48 in this fraction was assessed by the detection of a single band ( M r 260 000) after silver staining and Coomassie staining of 4–15% gradient SDS–PAGE. It was confirmed by the absence of apo B-100 contaminant in Western blot of the purified protein preparation. A linear relationship was observed between the densitometric analysis of SDS–PAGE bands and the apo B-48 in a protein range of 0–3 μg. In conclusion, preparative gel electrophoresis was used in a single step purification of apo B-48 that was adapted to the preparation of a standard solution.