nl5b01153_si_002.avi (1.79 MB)
Single Cell GFP-Trap Reveals Stoichiometry and Dynamics of Cytosolic Protein Complexes
mediaposted on 2015-05-13, 00:00 authored by Tim Wedeking, Sara Löchte, Christian P. Richter, Maniraj Bhagawati, Jacob Piehler, Changjiang You
We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level.