3.1. Methods An outline of the different methods is presented in Fig.13.1. The
Outline first step common to all these methods involves breaking cells in the presence of TCA, allowing thiol-acidic quenching, also resulting in the precipitation of soluble cellular proteins. From there, protein-thiol alkylation by IAM, NEM, or their derivatives or with other thiol-specific reagents is performed upon solubilizing TCA-precipitated proteins by increasing the pH of the solution to 7.5-9 and reducing oxidized thiols with DTT (see Note1). When applicable, differential alkylation of reduced- vs oxidized-cysteine residues is achieved, sequentially before and after reduction of oxidized residues using the thiol-reducing agents DTT or P-mercaptoethanol.
The following techniques all rely on the differential separation of oxidized vs reduced proteins by either sodium-dodecyl sulfate polyacrylamide gels electrophoresis (SDS-PAGE) or by isoelectrofocalization and on their visualization by western blot.
They are thus adapted for proteins for which specific antibodies are available. They can be also applied to purified proteins that are then visualized by gel Coomassie staining.
188.8.131.52. Disulfide-Linked In the simplest case, the presence of an intermolecular disulfide
Protein Complexes bond between the protein of interest and another known or unknown protein can be easily visualized with SDS-PAGE under nonreducing conditions (see Fig.13.2A,B). The disulfide-linked complex will migrate at a size corresponding approximately to the sum of the size of the proteins forming this complex. Comparative migration under nonreduced and reduced conditions will establish that the change of mobility of the protein of interest observed under nonreducing conditions is the consequence of a
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