Abstract
Cleavable Affinity Purification (Cl-AP) uses a tripartite system of Protein-A-Streptavidin beads and nanobodies, coupled with a biotinylated, thiol-cleavable linker, providing one-step affinity purification from lysates of tissues expressing tagged proteins. This technique allows fluorescent versions of mitotic protein complexes to be isolated intact from cells, for use in biophysical and microscopy-based assays, overcoming the traditional limitations of reductionist approaches. We have used this technique successfully to purify both GFP-tagged and mCherry-tagged proteins, and their interacting partners, expressed in Drosophila melanogaster embryos. Although we demonstrate the efficacy of the GFP-binding protein and RFP-binding protein nanobodies from Chromotek, in theory any antibody could be coupled to the beads and used as a Cl-AP reagent.
Highlights
[Background] Many proteins elicit their cellular function as part of multi-protein complexes
Purification of specific proteins or complexes from cells/tissues generally relies on either co-immuno-precipitation or purification of a tagged version of the protein of interest which has been introduced into cells, for example using haemagglutinin (HA), FLAG3 or tandem affinity purification (TAP)-tagging
There are two main issues with these in vivo approaches: (i) the beads themselves bind to non-specific proteins leading to contamination problems, (ii) the strong antibody-antigen or tag-matrix interaction cannot be disrupted without disrupting the interactions between protein complex subunits
Summary
[Background] Many proteins elicit their cellular function as part of multi-protein complexes. In which the antibody-antigen interactions are disrupted using peptide competition or in which the tag is cleaved using proteases can potentially result in the purification of soluble, intact protein complex from cells. Allowing the isolation of a protein complex of interest in soluble form, in the buffer of choice. Beckman tube (Beckman Coulter, catalog number: 343776) 3.
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