sodium-dodecyl-sulfate and Testicular-Neoplasms

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.

Topics: Animals; Carcinoma, Embryonal; Cell Line, Tumor; Cell Membrane Permeability; Chromatography, Gel; Cross-Linking Reagents; DNA-Binding Proteins; Homeodomain Proteins; Immunoprecipitation; Male; Mercaptoethanol; Mice; Nanog Homeobox Protein; Octamer Transcription Factor-3; Sodium Dodecyl Sulfate; Succinimides; Testicular Neoplasms

A rapid method for the isolation of intracytoplasmic A particles, the putative intracellular nucleoprotein cores of mouse mammary tumor virus (MTV), is presented. Spontaneous C3H/He mouse mammary tumors and transplantable mouse Leydig cell tumor were used as source material. Large aggregations of intracytoplasmic A particles were separated from cellular contaminants on discontinuous sucrose gradients and subsequently further purified by isopycnic banding in linear sucrose gradients. The purified particles were solubilized in sodium dodecyl sulfate, and the structural proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified mouse mammary tumor virions were solubilized, and their proteins were analyzed in the same way. Comparison of co-electrophoretic gels indicated a lack of correlation in the molecular size of the major structural proteins in intracytoplasmic A particles and MTV. The three major proteins of the A particles were calculated to be 80,000; 35,000; and 20,000 daltons. Five major polyacrylamide gel electrophoresis bands were obtained with purified MTV; these were 90,000; 69,000; 55,000; 37,000; and 27,500 daltons. These figures showed good correlation with those published for MTV by Nowinski et al. These results suggest the need for the reexamination of the current tenet that intracytoplasmic particles represent intracellular MTV nucleoprotein cores.

Topics: Animals; Centrifugation, Density Gradient; Cytoplasm; Electrophoresis, Polyacrylamide Gel; Female; Inclusion Bodies, Viral; Leydig Cell Tumor; Male; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Inbred C3H; Microscopy, Electron; Molecular Weight; Neoplasms, Experimental; Nucleoproteins; Peptides; Sodium Dodecyl Sulfate; Sucrose; Testicular Neoplasms; Viral Proteins