Coupling tandem affinity purification and quantitative tyrosine iodination to determine subunit stoichiometry of protein complexes.
Rapid protein purification methodologies, such as strategies involving the tandem affinity purification module, have resulted in the identification of a tremendous number of multisubunit protein complexes. Furthermore, in this modern genomic age, mass spectrometry methods are often coupled with affinity purification to identify the genes that encode each protein subunit. However, simple methodologies to determine the stoichiometry of individual subunits within a multisubunit complex have not received much attention. In this article we describe a procedure to rapidly and efficiently determine the stoichiometry of subunits within multisubunit complexes using a combination of tandem affinity purification and quantitative 125I labeling of subunit tyrosines.
Smith
Corey L
CL
Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Biotech II Suite 210, Worcester, MA 01605, USA.
Peterson
Craig L
CL
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011865:Radioisotope Dilution Technique; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D014443:Tyrosine