Detection of serum insulin-like growth factor binding proteins on western ligand blots by biotinylated IGF and enhanced chemiluminescence.
A novel procedure for the detection of IGF binding capacity of IGFBPs on Western ligand blots (WLB) was developed using biotinylated IGFs as probes. The biotinylated IGF-IGFBP complexes were visualized by streptavidin-horseradish peroxidase and enhanced chemiluminescence (ECL). The procedure was found to be faster and more efficient than the conventional method with iodinated IGFs. In normal human serum a predominant doublet at 38-42 kDa and five smaller bands at 35, 34, 30, 28 and 24 kDa were detected by both methods, whereas two additional bands at 26 and 16 kDa became visible with the ECL method. In pregnancy serum only one single faint band at 30 kDa could be detected by the iodinated method. In contrast, the ECL method revealed five other bands at 42, 34, 28, 26 and 16 kDa. Besides the 38-42 kDa doublet, the 30 and 16 kDa bands reacted strongly with anti-IGFBP-3 antibodies in Western immunoblotting (WIB) and therefore were related to IGFBP-3 fragments. The technical advantages of this ECL method include an extremely short exposure time to the radiographic film and a long stability of the probe. In addition, the ECL method is a non-radioactive method, making radioprotection and radioactive waste removal unnecessary.
Op De Beeck
L
L
Department of Pediatrics, University of Antwerp, Belgium.
Verlooy
J E
JE
Van Buul-Offers
S C
SC
Du Caju
M V
MV
D001710:Biotin; D015153:Blotting, Western / Q000379:methods*; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000097:blood*; D007334:Insulin-Like Growth Factor I; D008163:Luminescent Measurements; D011994:Recombinant Proteins
D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D001710:Biotin