Fig. 1.

Workflow for affinity purification of GFP-tagged complexes on a microfluidic device. A limited number of cells (in this paper we used 12,000 cells for proof-of-principle) expressing a GFP-tagged form of a protein of interest (PI) are lysed to obtain whole cell extracts that serve as pools of proteins. Test samples as well as the appropriate controls are loaded into a microfluidic chip which is preloaded with all buffers and reagents that are required during the IP and preparation of the samples for the LC–MS/MS. During the microfluidic run, columns of GFP-nanobody beads are packed in single reactors and subsequently washed before loading of the protein samples from the inlet onto the columns. Tagged macromolecular assemblies are retained on the GFP-nanobody bead columns and digested to peptides. Peptide eluates are recovered and prepared for mass spectrometry analysis, after which specific interactors are identified