A biotinylated derivative of the tetrapeptide inhibitor ASTD-FMK, which acts as a competitive inhibitor for the cleavage site of pro-EMAP II (Endothelial monocyte-activating polypeptide).
CAT No: HB00026
Biotin-ASTD-FMK is a synthetic peptide-based inhibitor that incorporates both a biotin moiety and a fluoromethyl ketone (FMK) functional group, specifically designed for research applications involving the study of protease activity and target identification. As a biotinylated peptide, it combines the high affinity of biotin-streptavidin interactions with the irreversible inhibitory properties of FMK, enabling precise labeling and functional interrogation of active-site cysteine proteases. The unique structural features of Biotin-ASTD-FMK make it a valuable tool for researchers seeking to investigate enzyme mechanisms, identify protease substrates, and facilitate the isolation of target proteins from complex biological samples.
Protease activity profiling: In biochemical research, Biotin-ASTD-FMK serves as a potent and selective probe for profiling cysteine protease activity in cell lysates, tissue extracts, or purified enzyme preparations. The FMK group covalently binds to the active site cysteine residue of target proteases, resulting in irreversible inhibition and stable enzyme labeling. The biotin tag enables subsequent detection or capture using streptavidin-based systems, allowing researchers to visualize, quantify, and compare protease activity under various experimental conditions. This application is particularly valuable in studies focused on elucidating protease function in physiological and pathological processes.
Affinity purification of proteases: The biotinylated nature of this inhibitor allows for efficient affinity purification of labeled proteases from complex biological mixtures. Upon covalent modification of target enzymes, the biotin tag facilitates their selective capture using streptavidin-conjugated beads or surfaces, streamlining the isolation and enrichment of active protease populations. This approach supports downstream analyses such as mass spectrometry, immunoblotting, or structural characterization, providing critical insights into enzyme identity, abundance, and post-translational modifications.
Substrate identification and inhibitor screening: Biotin-ASTD-FMK is instrumental in substrate trapping experiments, where its irreversible binding to active proteases enables the stabilization and subsequent identification of enzyme-substrate complexes. By applying this reagent in biochemical assays, researchers can capture transient interactions that are otherwise challenging to detect, thereby advancing the discovery of novel substrates and mapping of proteolytic pathways. Additionally, its use in competitive inhibition assays aids in the evaluation of new inhibitor candidates by assessing their ability to prevent probe labeling of target enzymes.
Cellular localization studies: The biotin tag incorporated into the inhibitor facilitates the visualization of labeled proteases within cellular or tissue contexts. Following treatment with the probe, streptavidin-conjugated fluorophores or enzymes can be used to detect and localize the distribution of active proteases via microscopy or histochemical staining. This capability supports investigations into the spatial regulation of protease activity and its role in diverse biological processes, including apoptosis, inflammation, and protein turnover.
Mechanistic enzymology research: Researchers utilize Biotin-ASTD-FMK to dissect the catalytic mechanisms of cysteine proteases by enabling the capture of covalently modified enzyme intermediates. The stable enzyme-inhibitor complexes generated by this reagent can be subjected to detailed kinetic, structural, or mutagenesis studies, providing mechanistic insights into substrate recognition, active site architecture, and the effects of specific mutations. Such applications are fundamental to advancing the understanding of protease biology and informing the rational design of selective inhibitors for research use.
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