Purification of protein complexes and characterization of protein-protein interactions.
In plants and all other multicellular organisms, both the intra- and extracellular environments are filled with dynamic biomolecular interactions that control many biological processes. Most of these interactions are biochemical in nature and often exist between proteins. For instance, many protein-protein interactions assist in sustained cellular homeostasis but also allow for rapid intracellular communication in response to stimuli. Thus, the discovery and validation of protein-protein interactions, and the consequent formation of protein complexes, is an integral and essential component of plant biology research. The ability to efficiently and accurately determine existing protein networks is necessary to further our understanding of plant biology. However, discovering protein networks represents a challenge for both present and future researchers. Here we have outlined several straightforward methods aimed at first discovering protein-protein interactions and then characterizing them utilizing additional approaches. We first describe methods for rate-zonal centrifugation, in vitro binding assays, and co-immunoprecipitation experiments in the context of discovering novel protein-protein interactions. Next, we discuss methods for characterizing and validating these interactions using alternative approaches: yeast two-hybrid, in vitro pull-down assays, and bimolecular fluorescence complementation (BiFC). Obviously each of these methods need not be performed in parallel; rather our goal was to describe several approaches, some of which may be more appropriate for increasingly specialized laboratory environments.
Department of Biochemistry, Life Sciences Center, University of Missouri, Columbia, MO, USA.
Kirby N
KN
Swatek
Chris B
CB
Lee
Jay J
JJ
Thelen
D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D047468:Immunoprecipitation; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D029681:Arabidopsis Proteins; D046912:Multiprotein Complexes