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Updated: Sep 1, 2026

Immunoblot Analysis
Published on: June 20, 2008
A rapid, non-radioactive method for the detection of insulin-like growth factor binding proteins by Western ligand
1Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Insulin-like growth factor binding proteins (IGFBPs) are produced by many cell types and are present in a wide variety of biological fluids. Detection of IGFBPs in complex fluids is commonly performed by Western ligand blotting using 12SI-IGF-I and/or 125I-IGF-II as the labeling ligand. This method is extremely useful in detecting IGFBPs; however, it requires the use of radioactive materials and usually takes several days to weeks to complete. Herein, we report on a modification of this method using biotinylated IGFs to detect both purified recombinant IGFBPs, as well as IGFBPs in fibroblast conditioned medium. This alternative method has proved sensitive, time- and cost-efficient, and it is environmentally safe.
Insulin-like growth factor binding proteins (IGFBPs) are produced by many cell types and are present in a wide variety of biological fluids. Detection of IGFBPs in complex fluids is commonly performed by Western ligand blotting using 12SI-IGF-I and/or 125I-IGF-II as the labeling ligand. This method is extremely useful in detecting IGFBPs; however, it requires the use of radioactive materials and usually takes several days to weeks to complete. Herein, we report on a modification of this method using biotinylated IGFs to detect both purified recombinant IGFBPs, as well as IGFBPs in fibroblast conditioned medium. This alternative method has proved sensitive, time- and cost-efficient, and it is environmentally safe.
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Western Blotting
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
Enzyme-Linked Immunosorbent Assay
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or quantified.

