Ligand blot analysis: validation of quantitative capabilities and utilization for measurement of truncated insulin-like growth factor regulation of Hep-G2 insulin-like growth factor binding protein-1 production.
The quantitative capabilities of Western "ligand" blot analysis (LBA) of insulin-like growth factor binding protein have been evaluated using iodine-labeled insulin-like growth factor-II (125I-IGF-II), purified amniotic fluid insulin-like growth factor binding protein-1 (IGFBP-1), and IGFBP-1 in conditioned medium (CM) from Hep-G2 cells. Optimal conditions were evaluated for analysis of IGFBP-1 using IGF-2-II as the ligand. IGFBP-1 ligand blot quantities were measured after autoradiography by one- (1D) and two (2D)-dimensional densitometry. The 2D method was found to exhibit higher linearity (IGF-I, r = 0.993; IGF-II, r = 0.984) over a wider range than the 1D method. Displacement curves for IGF-II against membrane-bound IGFBP-1 measured by LBA were similar to those obtained with IGFBP-1 solutions using antibody precipitation of IGFBP-1. Transfer of IGFBP-1 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels to nitrocellulose membranes was linear across the range 3.1-400 ng with both the 0.45- and the 0.2-micron pore sizes tested. The 0.2-micron size, however, showed nearly quantitative binding of IGFBP-1 trapping 20% more of this binding protein than the larger pore size. LBA was used to measure IGFBP-1 in CM from control and des1-3IGF-I (trIGF-I)-treated Hep-G2 cells and the results were compared with those obtained by a specific IGFBP-1 radioimmunoassay. IGFBP-1 concentrations measured by both assays were similar and both demonstrated nearly identical sensitivities. Each assay confirmed the previously noted trIGF-I suppression of IGFBP-1 secretion by Hep-G2 cells.
Grissom
F
F
Department of Endocrinology, Karolinska Institute, Stockholm.
Rivero-Crespo
F
F
Lindgren
B
B
Hall
K
K
D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1; D007335:Insulin-Like Growth Factor II / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D008114:Liver Neoplasms, Experimental / Q000737:chemistry; D008567:Membranes, Artificial; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018970:Insulin-Like Growth Factor Binding Protein 1; D008567:Membranes, Artificial; D007335:Insulin-Like Growth Factor II; D003101:Collodion