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Plasma membranes have integral transmembrane proteins involved in facilitated transport. These proteins are collectively referred to as transport proteins, and they function as either channels for the material or as carriers themselves. Channel proteins have hydrophilic domains exposed to the intracellular and extracellular fluids and a hydrophilic channel through their core that provides a hydrated opening for solutes to pass through the membrane layers. Passage through the channel allows...
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Detection of Detergent-sensitive Interactions Between Membrane Proteins
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Detection of Detergent-sensitive Interactions Between Membrane Proteins.

Nava Zaarur1, Xiang Pan2, Konstantin V Kandror2

  • 1Department of Biochemistry, Boston University School of Medicine; nzaarur@bu.edu.

Journal of Visualized Experiments : Jove
|March 27, 2018
PubMed
Summary

Researchers developed a new assay to study weak protein interactions, like between sortilin and glucose transporter GLUT4. This method is fast, inexpensive, and useful for analyzing detergent-sensitive interactions in cells.

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Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo
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Area of Science:

  • Cellular Biology
  • Biochemistry
  • Molecular Interactions

Background:

  • Understanding protein-protein interactions is crucial for deciphering cellular regulatory networks.
  • Detecting luminal interactions between membrane proteins, such as sortilin and glucose transporter GLUT4, presents experimental challenges due to detergent sensitivity, limiting traditional methods like co-immunoprecipitation.

Purpose of the Study:

  • To develop a rapid, cost-effective assay for validating weak, detergent-sensitive interactions between membrane proteins.
  • To specifically validate the interaction between the sorting receptor sortilin and glucose transporter GLUT4.

Main Methods:

  • Chemically synthesized a myc-tagged peptide representing the sortilin-binding epitope on GLUT4.
  • Expressed and purified histidine-tagged sortilin from mammalian cell lysates using Cobalt beads.
  • Immobilized sortilin on beads and incubated with the GLUT4 peptide at varying pH, followed by Western blot analysis of eluted material.

Main Results:

  • Successfully demonstrated the interaction between sortilin and the GLUT4-derived peptide using the developed assay.
  • The assay allowed for the analysis of binding under different pH conditions.

Conclusions:

  • The described assay provides a simple, fast, and inexpensive method for validating weak, detergent-sensitive protein-protein interactions.
  • This assay is adaptable for studying other challenging luminal interactions between membrane proteins.