Disulfide trapping

Changing the carbon disulfide trapping agent from copper acetate and diethylamine solution to methanolic potassium hydroxide to produce a xanthogenate increases the method s reproducibility and sensitivity. " ... 

Buck, E., Wells, J.A. Disulfide trapping to localize small-molecule agonists and antagonists for a G protein-coupled receptor. Proc. Natl Acad. Sci. USA 2005, 102, 2719-2724. 

As implemented by Sukharev and Guy, the combination of patch-clamp measurements with disulfide trapping provides a sensitive approach for testing models of the open state by characterizing the consequences of disulfide bond formation between specific cysteines on... 

Klco, J. M., Lassere, T. B., and Baranski, T. J. (2003). C5a receptor oligomerization. I. Disulfide trapping reveals oligomers and potential contact surfaces in a G protein-coupled receptor. J. Biol. Chem. 278, 35345-35353. 

Disulfide Trapping for Modeling and Structure Determination of ReceptorrChemokine Complexes... 

Coexpression of disulfide-trapped complexes between receptors and chemokines in insect Sf9 cells is similar to coexpression of noncovalent complexes and is described in detail by Gustavsson et al in Chapter 11. Briefly, the PI stocks of the two types of particles (those carrying receptor and those carrying chemokine) are used to coinfect Sf9 cells at a density of 2—2.6 X 10° ceUs/mL. Even if the multiphcity of infection (MOI) has been previously separately optimized for each vector, it frequently needs to be reoptimized in the context of the coexpression experiment, likely because the cell expression machinery gets taxed to a different degree when producing individual components versus the complex (Fig. 5). 

Table 1 Comparison of Experimental Approaches for Characterization of Disulfide-Trapped ReceptonChemokine Complexes... 

The result of a flow cytometry experiment is a histogram of cell distribution by the total amount of surface-bound chemokine, with or without an unlabeled competitor Hgand. This assay provides an easy way to quickly screen numerous candidate disulfide-trapped construct combinations uHth-out the complexities of sample purification however, the utUity of this method is limited to chemokines that are detectable on the cell surface by a... 

Quantification and Characterization of Disulfide-Trapped Compiexes by SDS-PAGE and Western Blotting... 

We usually conduct Western blot analysis on purified samples and interpret it in conjunction with other assays described herein SDS-PAGE, SEC, and DSF. However, unlike these other assays. Western blotting does not require the samples to be purified and thus can be conducted on whole cell lysates as an alternative first line of analysis to quickly screen for promising disulfide-trapped constructs. 

Characterization of Monodispersity and Thermal Stability of Disulfide-Trapped Complexes with SEC and CPM-DSF... 

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