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Related Experiment Videos

Biotinylinsulins as potential tools for receptor studies.

K Hofmann, F M Finn, H J Friesen

    Proceedings of the National Academy of Sciences of the United States of America
    |July 1, 1977
    PubMed
    Summary

    Researchers developed a method to attach insulin to Sepharose beads using biotin-avidin bonds. This modified insulin retains its ability to stimulate adipocytes, enabling targeted hormone immobilization for research applications.

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    Clock gene expression in the liver of streptozotocin-induced and spontaneous type 1 diabetic rats.

    Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme·2013

    Area of Science:

    • Biochemistry
    • Bioconjugation Chemistry
    • Hormone Research

    Background:

    • Affinity chromatography requires stable immobilization of biomolecules.
    • Biotin-avidin interactions offer a strong, specific, noncovalent binding system.
    • Targeted attachment of hormones to solid supports is crucial for various biological assays.

    Purpose of the Study:

    • To describe the preparation of insulin-affinity columns using biotin-avidin noncovalent bonds.
    • To evaluate the biological activity and binding characteristics of biotinylated insulin.
    • To demonstrate the utility of this method for creating functional hormone-immobilized supports.

    Main Methods:

    • Selective acylation of insulin at the B chain's N-terminus with biotin N-hydroxysuccinimido ester to create N(alpha,B1)-biotinylinsulin.

    Related Experiment Videos

  • Assessment of N(alpha,B1)-biotinylinsulin's ability to stimulate rat epididymal adipocytes.
  • Demonstration of N(alpha,B1)-biotinylinsulin's affinity for avidin via displacement assays and electrophoresis.
  • Immobilization of N(alpha,B1)-biotinylinsulin onto avidin-Sepharose to form affinity columns.

    • Main Results:

    • N(alpha,B1)-biotinylinsulin stimulated adipocytes with 94% of the potency of native insulin.
    • The biotinylated insulin formed a stable complex with avidin, visualized by electrophoresis.
    • Insulin-affinity columns prepared with biotinylinsulin on avidin-Sepharose retained biological activity.
    • The immobilized insulin on beads showed approximately 15% of the activity of free biotinylinsulin.

    Conclusions:

    • Biotinylation provides a versatile strategy for the targeted, noncovalent attachment of insulin to solid supports.
    • The resulting biotinylinsulin-avidin-Sepharose affinity columns are biologically active and suitable for research.
    • This approach offers advantages for preparing hormone-based affinity matrices for various applications.