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Recent genome sequencing projects have identified new peptidases in multiple organisms, many with unknown functions, suggesting the need for new tools to study these enzymes. This unit outlines selection and use of small-molecule and protein-based probes to covalently modify peptidases in complex cellular environments. These activity-based probes (ABPs) have been designed based on well characterized peptidase inhibitor scaffolds, but make use of new techniques to greatly enhance their utility for studying families of related peptidases. In particular, ABPs can be used to track activity of peptidases in crude cell extracts, intact cells, and in vivo, allowing rapid purification and identification of labeled targets. They can be used with libraries of small molecules to rapidly assess potency and selectivity of compounds in complex, physiologically relevant samples. Probe selection, probe tagging using reporters, labeling of recombinant targets, crude protein extracts, and peptidase targets in cell culture systems, affinity purification of targets, and inhibitor screening using affinity probes are outlined.

Original publication

DOI

10.1002/0471140864.ps2117s36

Type

Journal article

Journal

Curr Protoc Protein Sci

Publication Date

09/2004

Volume

Chapter 21

Pages

Unit - 21.17

Keywords

Fluorescent Dyes, Hydrolysis, Indicators and Reagents, Molecular Probes, Peptide Hydrolases, Proteins