sodium-dodecyl-sulfate and dithiobis(succinimidylpropionate)

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

Monoclonal antibodies that recognize cell surface proteins may serve as very useful tools for the study of the biological functions of membrane proteins. However, solubilization of the antigens with detergents may lead to major conformational changes of the protein, making their determination with monoclonal antibodies by immune blot or ordinary immunoprecipitation methods difficult. This is especially evident when the monoclonal antibodies recognize tertiary structures of the proteins in the membrane. We have generated two monoclonal antibodies which are specific for the cell surface antigens of multidrug-resistant human cell lines. However, the antigens of both monoclonal antibodies were difficult to detect by either immune blot or ordinary immunoprecipitation methods. We used a cleavable crosslinking reagent dithiobis(succinimidyl propionate) to covalently link the monoclonal antibody with its antigenic determinant in the membrane of intact cells. By this method, we were able to detect the antigens for these two monoclonal antibodies following solubilization, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method should have wide applicability in determination of membrane antigens recognized by monoclonal antibodies when immune blot or ordinary immunoprecipitation methods are not successful.

Topics: Antibodies, Monoclonal; Antigen-Antibody Complex; Antigens, Surface; Cell Line; Chemical Precipitation; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Humans; Leukemia; Sodium Dodecyl Sulfate; Solubility; Succinimides

Phosphatidylserine decarboxylase from Escherichia coli, an intrinsic membrane protein, catalyzes the conversion of phosphatidylserine to phosphatidylethanolamine. The physical and kinetic properties of the purified enzyme were studied in several detergents under assay conditions. The active form of the enzyme is an oligomer, probably a trimer, and the enzyme activity was unaffected by the concentration of the nonionic poly(oxyethylene) ether detergent present in the assay medium, so long as the detergent micelle/substrate mole ratio was less than one. When this ratio was greater than one, the detergent acted as an inhibitor by competing with enzyme-containing micelles for substrate. The zwitterionic and bile salt detergents that were tested inactivated the enzyme by dissociating the oligomer. The native, Triton X-100 solubilized, enzyme was modified with a cross-linking reagent. Activity of the cross-linked enzyme was retained after the Triton X-100 was replaced by a zwitterionic sulfobetaine detergent and conformed to the same kinetic model as with the poly(oxyethylene) ether detergents. The cross-linked enzyme was also active when solubilized by the bile salt detergents although the activity did not conform to any simple kinetic model. These data indicate that the oligomer is the active form of the enzyme under assay conditions and that certain nondenaturing detergents can inactivate this enzyme by dissociating the enzyme complex.

Topics: Animals; Carboxy-Lyases; Cattle; Deoxycholic Acid; Detergents; Disulfides; Escherichia coli; Octoxynol; Phosphatidylserines; Polyethylene Glycols; Sodium Dodecyl Sulfate; Succinimides; Surface-Active Agents