Solid-phase preparation of protein complexes (original) (raw)
2010, Journal of Molecular Recognition
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Methods of Enzymology. Immunoaffinity Purification of Mammalian Protein Complexes
Methods in Enzymology, 2003
This chapter aims to describe the generation of recombinant cells and the technique for purification of the complex. Identification of the interaction partners of a particular protein became a valuable way to gain insight in the physiological role of the protein. Many proteins appear to exist and function as stable multimeric protein complexes. For instance, the multimeric RNA polymerase II protein complex consists of 12 polypeptides. It transcribes DNA templates as a complex, whereas individual subunits have no such activity, thus making it impossible to predict “bona fide” functions of an RNA polymerase II from a single subunit. Likewise, if the protein of interest functions as a complex, it is crucial to purify it as a complex to determine what it does in the cell. The chapter also explores the most widely used system to identify intermolecular interactive partners of a particular protein of interest, the yeast two-hybrid system. The stably expressed exogenous proteins with epitope tags are integrated in the complexes when the complex is synthesized de novo. This chapter discusses such protein complexes purified rapidly by immunoprecipitation with antibodies against epitopes.
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