MMP12 Inhibitor Screening Assay Kit (Colorimetric) (ab139441)

Overview

  • Product name

    MMP12 Inhibitor Screening Assay Kit (Colorimetric)
    See all MMP12 kits
  • Detection method

    Colorimetric
  • Sample type

    Inhibitor compounds
  • Assay type

    Enzyme activity
  • Product overview

    Abcam's MMP12 Inhibitor Screening Assay Kit (Colorimetric) (ab139441) is a complete assay system designed to screen MMP12 inhibitors using a thiopeptide as a chromogenic substrate (Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5). The MMP cleavage site peptide bond is replaced by a thioester bond in the thiopeptide. Hydrolysis of this bond by an MMP produces a sulfhydryl group, which reacts with DTNB [5,5’-dithiobis(2-nitrobenzoic acid), Ellman’s reagent] to form 2-nitro-5-thiobenzoic acid, which can be detected by its absorbance at 412 nm (e=13,600 M-1cm-1 at pH 6.0 and above). The assays are performed in a convenient 96-well microplate format. 

  • Notes

    This kit is useful to screen inhibitors of MMP12, a potential therapeutic target. The MMP inhibitor NNGH is also included as a prototypic control inhibitor.

    Thiol inhibitors should not be used with this kit, as they may interfere with the colorimetric assay.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests
    96-well Clear Microplate (1/2 Volume) 1 unit
    Colorimetric Assay Buffer 1 x 20ml
    MMP Inhibitor 1 x 50µl
    MMP Substrate 1 x 50µl
    MMP12 Enzyme (Human, Recombinant) 1 x 14µl
  • Function

    May be involved in tissue injury and remodeling. Has significant elastolytic activity. Can accept large and small amino acids at the P1' site, but has a preference for leucine. Aromatic or hydrophobic residues are preferred at the P1 site, with small hydrophobic residues (preferably alanine) occupying P3.
  • Tissue specificity

    Found in alveolar macrophages but not in peripheral blood monocytes.
  • Sequence similarities

    Belongs to the peptidase M10A family.
    Contains 4 hemopexin-like domains.
  • Domain

    The conserved cysteine present in the cysteine-switch motif binds the catalytic zinc ion, thus inhibiting the enzyme. The dissociation of the cysteine from the zinc ion upon the activation-peptide release activates the enzyme.
  • Cellular localization

    Secreted > extracellular space > extracellular matrix.
  • Information by UniProt
  • Alternative names

    • HME
    • Macrophage elastase
    • Macrophage metalloelastase
    • Matrix metalloproteinase-12
    • ME
    • MME
    • MMP-12
    • Mmp12
    • MMP12_HUMAN
    see all

Images

  • Slope=V=4.85E-03 OD/min.

  • control slope = 5.08E-03 OD/min
    inhibitor slope = 4.82E-04 OD/min
    inhibitor % activity remaining = (4.82E-04/5.08E-03) x 100 = 9.49%

  • Activity of a control sample =
    (4.85E-03OD/min x 1E-04L)/(13,600M-1cm-1 x 0.5cm)=
    7.13E-11 mol/min at 37°C, 100?M thiopeptolide

     

     

Protocols

References

ab139441 has not yet been referenced specifically in any publications.

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