Biotin-ATAD-FMK features an ATAD tetrapeptide linked to a fluoromethyl ketone warhead and a biotin tag. The construct binds and covalently modifies proteases with affinity for alanine- and threonine-rich motifs. Researchers use it for affinity enrichment, active-site labeling, and kinetic profiling. Its multifunctional architecture facilitates sensitive biochemical detection.
CAT No: HB00029
Biotin-ATAD-FMK is a synthetic, biotinylated peptide-based inhibitor designed for advanced biochemical and proteomic research. Structurally, it features a fluoromethyl ketone (FMK) warhead, which forms a covalent bond with the active site cysteine residue of target proteases, and a biotin moiety that facilitates affinity-based detection and enrichment. This dual-functional design makes Biotin-ATAD-FMK highly valuable for studies requiring precise labeling, isolation, and profiling of enzyme activity within complex biological samples. Its unique combination of selective inhibitory activity and biotin tagging enables researchers to interrogate protease function and dynamics with high specificity and sensitivity, supporting a wide range of applications in basic and applied life sciences.
Enzyme activity profiling: Researchers utilize Biotin-ATAD-FMK to selectively label and inhibit active cysteine proteases in cell lysates, tissue extracts, or in vitro systems. The FMK group reacts irreversibly with the catalytic cysteine, allowing for the covalent capture of active enzyme populations. Following labeling, the biotin tag enables subsequent affinity purification using streptavidin-based matrices, facilitating downstream analysis by SDS-PAGE, Western blotting, or mass spectrometry. This approach provides a robust tool for mapping protease activity profiles and identifying target enzymes under various physiological or experimental conditions.
Target identification and validation: In chemical biology and drug discovery workflows, Biotin-ATAD-FMK serves as a mechanism-based probe for identifying direct binding partners of covalent inhibitors. By incorporating this labeled inhibitor into proteomic experiments, scientists can enrich and isolate proteins that interact specifically with the FMK moiety. The biotin affinity handle allows for efficient pull-down of labeled proteins, followed by identification via mass spectrometry. This application is instrumental in validating the selectivity of novel inhibitor compounds and elucidating off-target effects, thereby advancing the rational design of protease-targeted therapeutics.
Activity-based protein profiling (ABPP): The compound is widely employed in ABPP platforms to monitor dynamic changes in protease activities within complex proteomes. By labeling only the catalytically active subset of enzymes, Biotin-ATAD-FMK enables quantitative comparisons of enzyme activity across different biological states, such as disease versus normal conditions or in response to pharmacological modulation. The resulting activity profiles yield valuable insights into protease regulation, substrate specificity, and functional roles in cellular processes, supporting systems-level investigations in molecular biology and biochemistry.
Enzyme inhibitor screening: The biotinylated FMK inhibitor is an effective reagent for evaluating the potency and selectivity of candidate protease inhibitors. In competitive binding assays, test compounds are assessed for their ability to prevent Biotin-ATAD-FMK from labeling target enzymes, providing a direct measure of inhibitory activity. This approach streamlines the identification of high-affinity inhibitors and supports structure-activity relationship (SAR) studies by enabling rapid screening of compound libraries against specific protease targets.
Affinity enrichment and interactome analysis: Beyond protease profiling, the biotin tag on Biotin-ATAD-FMK facilitates the selective enrichment of enzyme complexes and interacting partners from biological samples. By capturing covalently modified proteins on streptavidin-coated beads, researchers can isolate not only the targeted proteases but also associated regulatory proteins and cofactors. Subsequent proteomic analysis of these enriched fractions reveals protein-protein interaction networks and functional modules linked to protease activity, advancing the understanding of cellular pathways and molecular mechanisms in health and disease.
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