Crosslinking methods purification and analysis of crosslinked dynein products.
Axonemal dyneins are multi-megadalton complexes which consist of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs). The configuration and interactions among the many components within the dynein complex are not fully understood. For initial investigation of protein-protein interactions, chemical crosslinking can be easily applied to either flagellar axonemes or purified dyneins. Careful selection of crosslinker enables one to target protein-protein interactions that are constitutive and also to identify alterations in the configuration of the complex. For example, when performed in the presence of nucleotide or ligands such as Ca(2+), it is possible to trap transient interactions under specific physiological condition. Here I first describe the preparation of a crosslinked sample and its analysis by electrophoresis and immunoblotting using antibodies raised against a target and candidate interaction proteins. Next, when an interaction partner cannot be simply identified by immunoblotting, a crosslinked product may be isolated by immunoprecipitation, and its composition determined by mass spectrometry. These general approaches have great potential to define protein-protein interactions within any macromolecular complex of interest.
Sakato
Miho
M
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
D000818:Animals; D054468:Axoneme / Q000737:chemistry; D002696:Chlamydomonas / Q000166:cytology; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004398:Dyneins / Q000737:chemistry* / Q000648:ultrastructure; D006801:Humans; D015151:Immunoblotting / Q000379:methods
D003432:Cross-Linking Reagents; D004398:Dyneins