MMP1 Inhibitor Screening Assay Kit (Colorimetric) (ab139443)
Key features and details
- Assay type: Enzyme activity
- Detection method: Colorimetric
- Platform: Microplate reader
- Sample type: Inhibitor compounds
Overview
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Product name
MMP1 Inhibitor Screening Assay Kit (Colorimetric)
See all MMP1 kits -
Detection method
Colorimetric -
Sample type
Inhibitor compounds -
Assay type
Enzyme activity -
Product overview
Abcam MMP1 Inhibitor Screening Assay Kit (Colorimetric) (ab139443) is a complete assay system designed to screen MMP1 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 (ε=13,600 M-1cm-1 at pH 6.0 and above). The assays are performed in a convenient 96-well microplate format.
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Notes
This kit is useful to screen inhibitors of MMP1, 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.
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Platform
Microplate reader
Properties
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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 MMP1 Enzyme (Human, Recombinant) 1 x 66µl -
Research areas
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Function
Cleaves collagens of types I, II, and III at one site in the helical domain. Also cleaves collagens of types VII and X. In case of HIV infection, interacts and cleaves the secreted viral Tat protein, leading to a decrease in neuronal Tat's mediated neurotoxicity. -
Sequence similarities
Belongs to the peptidase M10A family.
Contains 4 hemopexin-like domains. -
Domain
There are two distinct domains in this protein; the catalytic N-terminal, and the C-terminal which is involved in substrate specificity and in binding TIMP (tissue inhibitor of metalloproteinases).
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. -
Post-translational
modificationsUndergoes autolytic cleavage to two major forms (22 kDa and 27 kDa). A minor form (25 kDa) is the glycosylated form of the 22 kDa form. The 27 kDa form has no activity while the 22/25 kDa form can act as activator for collagenase. -
Cellular localization
Secreted > extracellular space > extracellular matrix. - Information by UniProt
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Alternative names
- 27 kDa interstitial collagenase
- CLG
- CLGN
see all
Images
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Plot of OD vs time
Slope = V = 1.20E-02 OD/min
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Inhibition of MMP1 by NNGH (1 µM)control slope = 1.20E-02 OD/min
inhibitor slope = 2.13E-03 OD/min
inhibitor % activity remaining = (2.13E-03/1.20E-02) x 100 = 17.7%
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Inhibition of MMP1 by NNGH (1 µM) -
Example graph for Km and Vmax determinationKm = 1410 µM
Vmax = 40.0 pmol/sec
