Methods for identification of centrosome-associated proteins

Abstract

The centrosome has been analyzed by employing various strategies of genetics, cell biology, immunology, and protein biochemistry. This chapter outlines approach for the identification of centrosome associated proteins. This strategy includes four major steps: (1) isolation and purification of centrosomes and centrosome-containing structures; (2) preparation of anticentrosomal antibodies; (3) molecular cloning of DNA by immunoscreening of expression libraries; and (4) evaluation of the specificity of encoded proteins to the centrosome by the generation of fusion protein antibodies and protein expression by transient transfection. Although each step is considered to be labor-intensive and time-consuming, rapid advancement in antibody technologies and molecular cloning techniques has made it possible to carry out experiments in a simple and straightforward manner. This chapter therefore focuses on the third and fourth steps for molecular cloning and characterization of cDNA. This approach has allowed identifying unknown molecular components of the centrosome. It is essential to confirm that cDNA clones encode proteins localized at and/or around the centrosome. The two methods used for the evaluation of the specificity of cDNA clones include generation of bacterial fusion protein antibodies and protein expression in mammalian cells by transient transfection.