Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells

Jonghwan Kim; Alan B. Cantor; Stuart H. Orkin; Jianlong Wang

doi:10.1038/nprot.2009.23

Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells

Jonghwan Kim, Alan B. Cantor, Stuart H. Orkin, Jianlong Wang

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117 Scopus citations

Abstract

In gene regulation, proteins function as members of protein complexes to recognize chromosomal target DNA loci. In dissecting the pluripotent state in mouse embryonic stem (mES) cells, we have used in vivo biotinylation of critical transcription factors for affinity purification of protein complexes and chromatin immunoprecipitation (ChIP)-on-chip for target identification, respectively. Here, we describe detailed procedures for such studies to dissect protein-protein and protein-DNA interactions in mES cells. Specifically, the following three procedures will be described: (i) in vivo biotinylation system setup in mES cells; (ii) affinity purification of multiprotein complexes by one-step streptavidin capture and tandem anti-FLAG/streptavidin affinity purification; (iii) biotin-mediated ChIP (bioChIP). The system setup takes ∼50 d to complete, and it takes another ∼15 d and ∼3 d to perform affinity purification of protein complexes and bioChIP, respectively.

Original language	English
Pages (from-to)	506-517
Number of pages	12
Journal	Nature Protocols
Volume	4
Issue number	4
DOIs	https://doi.org/10.1038/nprot.2009.23
State	Published - 2009
Externally published	Yes

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title = "Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells",

abstract = "In gene regulation, proteins function as members of protein complexes to recognize chromosomal target DNA loci. In dissecting the pluripotent state in mouse embryonic stem (mES) cells, we have used in vivo biotinylation of critical transcription factors for affinity purification of protein complexes and chromatin immunoprecipitation (ChIP)-on-chip for target identification, respectively. Here, we describe detailed procedures for such studies to dissect protein-protein and protein-DNA interactions in mES cells. Specifically, the following three procedures will be described: (i) in vivo biotinylation system setup in mES cells; (ii) affinity purification of multiprotein complexes by one-step streptavidin capture and tandem anti-FLAG/streptavidin affinity purification; (iii) biotin-mediated ChIP (bioChIP). The system setup takes ∼50 d to complete, and it takes another ∼15 d and ∼3 d to perform affinity purification of protein complexes and bioChIP, respectively.",

author = "Jonghwan Kim and Cantor, {Alan B.} and Orkin, {Stuart H.} and Jianlong Wang",

year = "2009",

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T1 - Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells

AU - Kim, Jonghwan

AU - Cantor, Alan B.

AU - Orkin, Stuart H.

AU - Wang, Jianlong

PY - 2009

Y1 - 2009

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AB - In gene regulation, proteins function as members of protein complexes to recognize chromosomal target DNA loci. In dissecting the pluripotent state in mouse embryonic stem (mES) cells, we have used in vivo biotinylation of critical transcription factors for affinity purification of protein complexes and chromatin immunoprecipitation (ChIP)-on-chip for target identification, respectively. Here, we describe detailed procedures for such studies to dissect protein-protein and protein-DNA interactions in mES cells. Specifically, the following three procedures will be described: (i) in vivo biotinylation system setup in mES cells; (ii) affinity purification of multiprotein complexes by one-step streptavidin capture and tandem anti-FLAG/streptavidin affinity purification; (iii) biotin-mediated ChIP (bioChIP). The system setup takes ∼50 d to complete, and it takes another ∼15 d and ∼3 d to perform affinity purification of protein complexes and bioChIP, respectively.

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Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells

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