An Efficient Fluorescent Protein-Based Multifunctional Affinity Purification Approach in Mammalian Cells
Knowledge of an individual protein’s modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501–511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.
Key wordsMultifunctional affinity purification (MAP) TAP Fluorescent protein Venus FACS Protein complex MudPIT LC-MS/MS Proteomics
This work was supported by NIH grant GM068786 to D.M., NCI T32CA119925 to J.R.M., and Howard Hughes Medical Institute for K.L.G.
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