An Improved In Vivo Biotinylation Strategy Combined with FLAG and Antibody Based Approaches for Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells
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The proteome in mouse embryonic stem cells has not been extensively studied in comparison to other cellular systems, limiting our understanding of multi-protein complex functions in stem cell biology. Several affinity purification techniques followed by mass spectrometry analysis have been designed and validated to identify protein–protein interaction networks. One such approach relies on in vivo biotinylation of a protein of interest and subsequent pull-down of its interacting partners using streptavidin-conjugated agarose beads. This technique takes advantage of the high affinity between biotin and streptavidin, allowing for high affinity purification of protein complexes without the use of antibodies. Here, we describe an improved large-scale purification of multi-protein complexes in mouse embryonic stem cells by in vivo biotinylation, complemented with standard antibody and/or FLAG based affinity captures. This combined strategy benefits from the high efficiency of the streptavidin pull-down and the validation of the most highly confident interacting partners through the two alternative approaches.
Key wordsESC Biotinylation Streptavidin Multi-protein complexes Purification Mass spectrometry BirA
This research was funded by grants from the National Institutes of Health (NIH) to J.W. (1R01-GM095942) and the Empire State Stem Cell Fund through New York State Department of Health (NYSTEM) to J.W. (C026420, C028103, C028121). J.W. is also a recipient of Irma T. Hirschl and Weill-Caulier Trusts Career Scientist Award.
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