Biotin tagging activity-based probes

Activity-based probes (ABPs) are tagged covalent and irreversible enzyme inhibitors. Formation of a stable covalent bond ensures that the inhibitor will remain attached to the polypeptide after protein denaturation, after which the tag (radio-isotope, biotin, fluorophore) allows visualization and/or identification of the thus modified enzyme or enzyme family. The first proteasome ABP described comprised a tritium-labeled lactacystin analog [6,7]. Proteasome bands are visualized on one-dimensional SDS-PAGE (sodium docecylsulfate-polyacrylamide gel electrophoresis) gel in an autoradiogram after treatment with a radiolabeled... 

Affinity Tags. One of the primary applications for activity-based probes is the direct isolation of labeled target proteins. Therefore, many of the commonly used activity-based probes contain tags for enrichment of labeled proteins using affinity resins. The most commonly used affinity tag is biotin, which binds to avadin resins with diffusion-limited kinetics. This high affinity is particularly important when probes are used to label low... 

Figure 11 Mechanism-based probes for cysteine protease active site labeling, (a) E-64-mediated inhibition of cysteine proteases via nucleophilic attack of the catalytic cysteine at the epoxy group, (b) E-64-based probe with biotin reporter tag and tyrosine as 125l iodination site for radioactive labeling, (c) Acyloxymethylketone probe for the complementary labeling of cysteine proteases.

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