Non-reduced SDS-PAGE detection by western-blot

Streptavidin affinity purification

Autoradiography Fluorimetry

Coomassie staining MS analysis

Fig. 13.1. Schematic overview of the different methods presented in this chapter.

(see Fig.13.1). Basically, the methods presented rely for most of them on the principle of sequential labeling of reduced versus oxidized cysteine residues. Cysteine-spedfic reagents used for labeling thiols are derived from the alkylating agents iodoacetamide (IAM)

or N-ethylmaleimide (NEM) and include the high molecular weight alkylating agents AMS and Mal-PEG, 1 4C-radiolabeled-(14, 15) and fluorescent-IAM and -NEM (16, 17). The thiol-reagent HPDP attaching to cysteine residues through a disulfide linkage is also commonly used in fusion with a biotin moiety (18). The redox forms that are accessible to analysis by these techniques are disulfide bonds, whether intra or intermolecular, including S-thiolation and the sulfinic and sulfonic acid forms. Cysteine residues in the sulfenic acid form are very difficult to identify because of their very unstable nature, although some researchers have succeeded in this task, relying either on the exclusive reduction of the sulfenic acid by sodium arsenite (19) or on its reaction with specific chemicals such as dimedone (20).

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