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Cold Spring Harb Protoc. 2019 Jun 3;2019(6). doi: 10.1101/pdb.prot098384.

INTACT Proteomics in Xenopus.

Wasson L1,2, Amin NM2,3, Conlon FL4,2,3.

Author information

1
Department of Genetics, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599.
2
University of North Carolina McAllister Heart Institute, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599.
3
Department of Biology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599.
4
Department of Genetics, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599; Frank_Conlon@med.unc.edu.

Abstract

Analysis of the molecular mechanisms driving cell specification, differentiation, and other cellular processes can be difficult due to the heterogeneity of tissues and organs. Therefore, it is critical to isolate pure cell populations in order to properly assess the function of certain cell types in the context of a tissue. This protocol describes use of the INTACT (isolation of nuclei tagged in specific cell types) method in Xenopus, followed by proteomics analysis of nuclear protein complexes. The INTACT protocol utilizes two transgenes: (1) a three-part nuclear targeting fusion (NTF) consisting of a nuclear envelope protein (Nup35) that targets the NTF to the nuclear membrane, an enhanced green fluorescent protein (EGFP) cassette for NTF visualization in live animals, and a biotin ligase receptor protein (BLRP) that provides a substrate for the biotinylation of the NTF, and (2) the E. coli ligase BirA (which biotinylates the NTF) tagged to mCherry (for visualization). Either or both transgenes are driven by a tissue-specific promoter, making this protocol easily adaptable to proteomics analyses of immunoprecipitated complexes from INTACT-isolated nuclei of multiple tissue types to determine the composition of protein complexes in pure cell populations.

PMID:
30150318
DOI:
10.1101/pdb.prot098384

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