Sodium dodecyl sulphate gel electrophoretic preparation of protein standard human apolipoprotein B-48.
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 microg. 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.
Saviana
B
B
Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, Equipe Mixte INSERM 00-14, Faculté de Médecome de Nancy, Vandoeuvre lès-Nancy, France.
Pons
L
L
Namour
F
F
Quilliot
D
D
Ziegler
O
O
Guéant
J L
JL
D000368:Aged; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000302:isolation & purification*; D001202:Ascitic Fluid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D012967:Sodium Dodecyl Sulfate
D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D012967:Sodium Dodecyl Sulfate