Successful co-immunoprecipitation of Oct4 and Nanog using cross-linking.
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.
Stem Cell Institute, Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota, Room 2-216, MTRF, 2001-6th Street SE, Minneapolis, MN 55455, USA.
Liying
L
Zhang
Samuel
S
Rayner
Nobuko
N
Katoku-Kikyo
Liudmila
L
Romanova
Nobuaki
N
Kikyo
D000818:Animals; D018236:Carcinoma, Embryonal / Q000378:metabolism; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000302:isolation & purification* / Q000378:metabolism; D047468:Immunoprecipitation; D008297:Male; D008623:Mercaptoethanol / Q000378:metabolism; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000302:isolation & purification* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013388:Succinimides / Q000494:pharmacology*; D013736:Testicular Neoplasms / Q000378:metabolism
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; D013388:Succinimides; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; C011240:dithiobis(succinimidylpropionate)