IP-FCM: Immunoprecipitation Detected by Flow Cytometry

Watch video protocols on JoVE - www.jove.com. Journal of Visualized Experiments (JoVE) is a scientific journal publishing video articles on biological experiments filmed at leading research institutions. JoVE is the first video journal indexed in PubMed. To see more of this protocol go to www.jove.com IP-FCM: Immunoprecipitation Detected by Flow Cytometry This video protocol was filmed at the Mayo Clinic. Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

Channel: YouTube Video Length: 42s Date Found: Fri, Mar 25 2011 1:25 PM Category: Technology Date Produced: Fri, Mar 25 2011 11:12 AM View Count: 0