JoVE Immunology and Infection
1Department of Pediatrics, Centre for Understanding and Preventing Infection in Children, University of British Columbia
Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.
Published October 15, 2013. Keywords: Immunology, Bacteria, Aerobic, Gram-Negative Bacteria, Immune System Diseases, Respiratory Tract Diseases, Burkholderia, proteins, glass beads, 2-D gel electrophoresis
1Research and Development, GE Healthcare Bio-Sciences AB
A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan™ DIGE technology.
Published November 26, 2008. Keywords: Biochemistry, Cell surface protein labelling, Ettan DIGE, CyDye DIGE Fluor minimal dyes, cell surface proteins, receptors, fluorescence, 2-D electrophoresis
1Biological Medical Research Center (BMFZ), University Duesseldorf
This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.
Published August 3, 2009. Keywords: Basic Protocols, protein in-gel detection, Coomassie G-250, colloidal staining, 1- and 2-dimensional SDS-gels
JoVE Clinical and Translational Medicine
1Center for Proteomics, Smith College, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Chemistry, Smith College, 4Department of Biological Sciences and Center for Proteomics, Smith College
A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.
Published December 14, 2011. Keywords: Medicine, skeletal muscle, gender, 2-D gel electrophoresis, HPLC/MS, mouse
JoVE Immunology and Infection
1Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität
This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.
Published June 19, 2012. Keywords: Infection, Immunology, amoeba, Dictyostelium discoideum, density gradient centrifugation, effector protein, Icm/Dot type IV secretion system, immuno-magnetic separation, Legionella pneumophila, macrophage, pathogen vacuole