Strep-Protein Interaction Experiment

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The Strep-Protein Interaction Experiment ( SPINE ) is a biochemical method for the detection of protein-protein interactions .

properties

The SPINE is a variant of the pulldown assay in which the contact between two or more proteins is detected by protein purification using a Strep tag on a known, mostly recombinant protein. The binding proteins are also purified through the purification. The purification is carried out by affinity chromatography (e.g. streptactin ) with a reversible cross-linking of the protein complexes by formaldehyde to fix the mostly temporary bond. The cross-linking is removed by boiling the cross-linked protein complexes in sample buffer . Further separation is then achieved by SDS-PAGE . The identification of the binding proteins is achieved by a subsequent in-gel digestion and analysis by mass spectrometry . The method was adapted for membrane proteins in order to reduce unspecific interactions due to the hydrophobic effect .

Individual evidence

  1. ↑ C. Herzberg, LA Weidinger, B. Dörrbecker, S. Hübner, J. Stülke, FM Commichau: SPINE: a method for the rapid detection and analysis of protein-protein interactions in vivo. In: Proteomics. Volume 7, number 22, November 2007, pp. 4032-4035, doi : 10.1002 / pmic.200700491 . PMID 17994626 .
  2. ↑ VS Müller, PR Jungblut, TF Meyer, S. Hunke: Membrane-SPINE: an improved method to identify protein-protein interaction partners of membrane proteins in vivo. In: Proteomics. Volume 11, number 10, May 2011, pp. 2124-2128, doi : 10.1002 / pmic.201000558 . PMID 21472855 .
  3. ↑ VS Müller, K. Tschauner, S. Hunke: Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo. In: Journal of visualized experiments: JoVE. Number 81, 2013, S., doi : 10.3791 / 50810 . PMID 24300168 .