2 articles published in JoVE
Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry Mohan Babu1,2, Olga Kagan1, Hongbo Guo1, Jack Greenblatt1,3, Andrew Emili1,3 1Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 2Deparment of Biochemistry, Research and Innovation Centre, University of Regina, 3Department of Medical Genetics and Microbiology, University of Toronto Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays Alla Gagarinova*1, Mohan Babu*2,3, Jack Greenblatt1,2, Andrew Emili1,2 1Department of Molecular Genetics, University of Toronto, 2Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 3Department of Biochemistry, Research and Innovation Centre, University of Regina Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.