Not all insulin-like growth factor-binding proteins (IGFBPs) are detectable by western ligand blotting: case studies of PC12 pheochromocytoma and rat anterior pituitary IGFBPs and proteolyzed IGFBP-3.
We studied the limitations of the Western ligand blot (WLB) for detecting insulin-like growth factor-binding proteins (IGFBPs). PC12 rat pheochromocytoma cells and rat anterior pituitary cells (AP) secrete IGFBPs that cannot be detected by WLB. We used affinity labeling, WLB, dot blotting, competitive binding, ion exchange chromatography, and deglycosylation to characterize these IGFBPs. These IGFBPs were compared with pregnancy protease-derived IGFBP-3 fragments that also bind insulin-like growth factors (IGFs), but are not detectable by WLB. We showed that PC12 IGFBP is cationic, not glycosylated, with 25,500 mol wt reduced (18,500 unreduced), with high affinity for IGF-II and low affinity for IGF-I. It cannot be detected by WLB and is not a proteolytic derivative of other IGFBPs or IGF-II receptors. Its binding activity is not destroyed by sodium dodecyl sulfate (SDS) and heating. It binds to nitrocellulose and IGF-II after dot blotting, but not to IGF-II during WLB. AP also secrete an IGFBP(s) that was not detectable by WLB. AP IGFBPs, unlike those of PC12, have a higher mol wt, and at least one component is glycosylated. The failure of WLB to detect these proteins remains unexplained. Pregnancy protease-derived IGFBP-3 fragments also bind IGFs and are not detectable by WLB. However, they do electrotransfer to nitrocellulose. The failure of WLB to detect these fragments is probably due to proteolysis rendering the binding site susceptible to irreversible denaturation (under conditions of WLB) during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that WLB, while valuable, may have significant limitations in specific cases. Other techniques must complement WLB for detection of IGFBPs in conditioned media and other biological specimens.
Ocrant
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Department of Pediatrics, Brown University, Providence, Rhode Island.
Fay
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CT
Pham
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Rosenfeld
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D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004563:Electrochemistry; D010450:Endopeptidases / Q000378:metabolism; D006031:Glycosylation; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D016716:PC12 Cells; D010446:Peptide Fragments / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000737:chemistry*; D051381:Rats
D000345:Affinity Labels; D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D010446:Peptide Fragments; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D010450:Endopeptidases