Single-Step Affinity Purification of ERK Signaling Complexes Using the Streptavidin-Binding Peptide (SBP) Tag
Elucidation of biological functions of signaling proteins is facilitated by studying their protein–protein interaction networks. Affinity purification combined with mass spectrometry (AP-MS) has become a favorite method to study protein complexes. Here we describe a procedure for single-step purification of ERK (Rolled) and associated proteins from Drosophila cultured cells. The use of the streptavidin-binding peptide (SBP) tag allows for a highly efficient isolation of native ERK signaling complexes, which are suitable for subsequent analysis by mass spectrometry. Our analysis of the ERK interactome has identified both known and novel signaling components. This method can be easily adapted for SBP-based purification of protein complexes in any expression system.
Key wordsStreptavidin-binding peptide SBP Affinity purification Mass spectrometry ERK Drosophila
This protocol was developed with participation of Manuel Valdes, Marla Tipping, and Wenjian Xu. The authors thank Heya Zhao for helpful comments on the manuscript. A.V. was supported by the NIH grant GM105813. L.Y. was supported by the UMass Boston Sanofi Genzyme Doctoral Fellowship. Mass spectrometry was performed at the Taplin Mass Spectrometry Facility at Harvard Medical School.
- 23.Yang L, Veraksa A (2015) SAINT output for a complete ERK-SBP purification dataset. https://xythos.umb.edu/xythoswfs/webui/_xy-e3032387_1-t_4JUTz7Bi. Accessed 10 Dec 2015