Ub-PA (Ubiquitin Activity-Based Probe)

Product Overview

Ub-PA (Ubiquitin Activity-Based Probe) is a chemical probe tool designed on the ubiquitin scaffold. Its C-terminus carries an electrophilic warhead (Propargylamide, PA) that specifically and irreversibly reacts with the catalytic cysteine residue in the active site of DUB enzymes, enabling a streamlined "capture-label-identify" workflow. Unlike fluorogenic substrates, Ub-PA is not a substrate for activity detection — it is a probe tool for covalently labeling and enriching DUB proteins themselves.

Mechanism of Action

Ub-PA mimics the natural C-terminal structure of ubiquitin and is recognized and bound by DUB enzymes at their active site. The electrophilic warhead (Propargylamide) then undergoes nucleophilic addition with the catalytic cysteine thiol group, forming a stable covalent thioether bond that irreversibly labels the active form of the DUB. The labeled DUB-probe complex can be detected by SDS-PAGE / Western Blot or enriched via biotin/fluorophore tags for MS-based identification.

Applications

• Endogenous DUB activity profiling (DUBome profiling) in cells
• Activity-state identification of specific DUB family members
• Cellular target engagement validation for DUB inhibitors
• Screening for DUB activity dysregulation in disease tissues
• Functional labeling via Click Chemistry for downstream readouts

Product Highlights

• High selectivity: labels only active DUBs; no cross-reactivity with inactive enzymes or other cysteine proteases
• Covalent binding: irreversible labeling compatible with denaturing MS sample preparation
• Excellent compatibility: compact PA warhead preserves ubiquitin scaffold recognition
• Alkyne handle for CuAAC Click Chemistry introduction of biotin or fluorophore tags

Experimental Tips

Recommended probe concentration: 1–5 μM. Labeling conditions: 37°C, 30–60 min. Avoid free-thiol reducing agents (e.g., β-ME) in lysis buffer; use TCEP instead. Labeled samples can be separated by SDS-PAGE and detected using anti-ubiquitin antibody (e.g., FK2) or streptavidin-HRP after Click Chemistry biotinylation.