Cysteine biotinylation

Bogy o s group developed several ABPs for cysteine proteases with epoxide, vinyl sulfone, and acyloxymethyl ketone reactive groups [42,43]. Particularly noteworthy is DCG04 (4), a biotinylated papain-family protease inhibitor with an epoxide reactive group that targets... 

Grunewald, M., Bendahan, A., and Kanner, B. I. (1998) Biotinylation of single cysteine mutants of the glutamate transporter GLT-1 from rat brain reveals its unusual topology. Neuron 21, 623-632. 

Streptavidin may be immobilized to a surface using methods used with other proteins. For instance, strept(avidin) can be adsorbed onto a number of surfaces and still retain the ability to bind biotin and biotinylated molecules. Strept(avidin)-coated magnetic beads, plastic combs, and silicon chips used in many applications are made in this manner. Alternatively, streptavidin may be covalently bound to form an oriented monolayer using a cysteine derivative [101]. 

The same authors also examined the S-nitrosylation of proteins by nNOS, in neuronal tissue from mice (Figure 11.10). Conversion of nitrosylated to biotinylated cysteines was done as above. Biotinylation was, in this case, used to extract the (formerly) nitrosylated proteins from the total mixture of cellular proteins by selective binding to solid-phase attached streptavidin. After retrieving the bound material by reduction with excess free thiol, the samples were run on a gel, blotted, and individual proteins detected using an-... 

Activity-based prohling of active cysteine proteases identihed cathepsin L as a proenkephalin processing protease in secretory vesicles. The activity probe DCG-04, the biotinylated form of E64c that inhibits cysteine proteases, was used for specihc affinity labeling of the 27 kDa protease enzyme of the prohormone thiol protease (FTP) complex (18, 22), which represents... 

The active caspase ELISAs quantify the events closely following the release of cytochrome c from mitochondria. The active caspase ELISA quantifies a single active caspase family member (19). The procedure uses the covalent modification of the active site cysteine with a biotinylated inhibitor to label active caspases and selected capture on a plate coated with a caspase-specific monoclonal antibody (MAb). In this chapter, a typical characterization of an active caspase ELISA is included as a demonstration of proof that the ELISA quantifies a specific active caspase. The ability of the assay to... 

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