Successful co-immunoprecipitation of Oct4 and Nanog using cross-linking

Liying Zhang, Samuel Rayner, Nobuko Katoku-Kikyo, Liudmila Romanova, Nobuaki Kikyo

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

The transcription factors Oct4 and Nanog are essential for the maintenance of an undifferentiated and pluripotent state in early embryonic cells, embryonic stem cells and embryonal carcinoma cells in humans and mice. These factors are co-localized to promoters of more than 300 genes, and synergistically regulate their activities. Currently, the molecular interaction between these two factors has not been well-characterized. During attempts to co-immunoprecipitate Oct4 and Nanog we found that cross-linking with dithiobis[succinimidylpropionate] was necessary to maintain their interaction. This result was supported by gel filtration analysis. Surprisingly, formaldehyde, a cross-linker commonly used during chromatin immunoprecipitation of Oct4 and Nanog, did not preserve the complex. Our findings demonstrate the effectiveness of using DSP to mitigate the instability of the interaction between these two particular proteins. Additionally, this solution may potentially allow us to identify novel members of the Oct4-Nanog complex, leading to better understanding of the regulatory mechanisms behind pluripotency.

Original languageEnglish (US)
Pages (from-to)611-614
Number of pages4
JournalBiochemical and Biophysical Research Communications
Volume361
Issue number3
DOIs
StatePublished - Sep 28 2007

Bibliographical note

Funding Information:
This research was supported in part by the National Institutes of Health Grant R01 GM068027 to N.K.

Keywords

  • Cross-linking
  • Dithiobis[succinimidylpropionate] (DSP)
  • Embryonal carcinoma cells
  • Embryonic stem cells (ES cells)
  • Formaldehyde
  • Gel filtration
  • Immunoprecipitation
  • Nanog
  • Oct4 and pluripotency

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