MMP Activity Assay Kit (Fluorometric - Red) (ab112147)

Overview

  • Product nameMMP Activity Assay Kit (Fluorometric - Red)
    See all MMP kits
  • Sample type
    Cell culture extracts, Adherent cells, Suspension cells, Purified protein
  • Assay typeDirect
  • Product overview

    ab112147 uses a fluorescence resonance energy transfer (FRET) peptide as a MMP substrate. In the intact FRET peptide, the fluorescence of one part is quenched by the other. Upon cleavage into two separate fragments by MMPs, the fluorescence is recovered. ab112147 is designed to check the general activity of a MMP enzyme. It can also be used to screen MMP inhibitors when a purified MMP enzyme is used.

    With excellent fluorescence quantum yield and longer wavelength, this substrate shows less interference from autofluorescence of test compounds and cellular components and is much more sensitive than an EDANS/Dabcyl FRET substrate. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 540/590 nm. The pH-independent fluorescence makes the assay reading available for the whole physiological pH range. The high photostability of this FRET peptide provides a useful imaging probe. Many labs have used this kit for the high throughput screening of MMP inhibitors as potential anticancer drug candidates. This assay might be also used for monitoring cancer cells.

    Visit our FAQs page for tips and troubleshooting.

  • Notes

    ab112147 should be stored Desiccated

  • Tested applicationsSuitable for: Functional Studiesmore details

Properties

    Applications

    Our Abpromise guarantee covers the use of ab112147 in the following tested applications.

    The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

    Application Abreviews Notes
    Functional Studies Use at an assay dependent concentration.

    MMP Activity Assay Kit (Fluorometric - Red) images

    • Tissues were lysed with RIPA buffer and activated with 2 mM APMA (1:1) for 1,2 and 3 hours at 37°C. Samples were then diluted to 5 mg/mL and 10 mg/mL with Assay Buffer. 50 µl of MMP containing sample was mixed with MMP Red Substrate. The fluorescence signal was monitored 30 min after the start of the reaction by using a microplate reader with a filter set of Ex/Em = 540/595 nm. The reading from all wells was subtracted with the reading from substrate control, which contains MMP Red Substrate but no MMPs.

    • Tissues were lysed with RIPA buffer and activated with 2 mM APMA (1:1) for 1,2 and 3 hours at 37°C. Samples were then diluted to 5 mg/mL and 10 mg/mL with Assay Buffer. 50 µl of MMP containing sample was mixed with MMP Red Substrate. The fluorescence signal was monitored 30 min after the start of the reaction by using a microplate reader with a filter set of Ex/Em = 540/595 nm. The reading from all wells was subtracted with the reading from substrate control, which contains MMP Red Substrate but no MMPs.

    • MMPs were activated with 2 mM APMA (1:1). Samples were then diluted to 0.6 µg/mL (30 ng per well) with Assay Buffer. 50 µl of MMP containing sample was mixed with MMP Red Substrate. The fluorescence signal was monitored 30 min after the start of the reaction by using a microplate reader with a filter set of Ex/Em = 540/595 nm. The reading from all wells was subtracted with the reading from substrate control, which contains MMP Red Substrate but no MMPs.

    • MMPs were activated with 2 mM APMA (1:1). Samples were then diluted to 0.6 µg/mL (30 ng per well) with Assay Buffer. 50 µl of MMP containing sample was mixed with MMP Red Substrate. The fluorescence signal was monitored 30 min after the start of the reaction by using a microplate reader with a filter set of Ex/Em = 540/595 nm. The reading from all wells was subtracted with the reading from substrate control, which contains MMP Red Substrate but no MMPs.

    • APMA-activated purified MMPs (30 ng each) were mixed with Red Substrate. The fluorescence signal was monitored one hour after the start of the reaction by using a microplate reader with a filter set of Ex/Em = 540/590 nm. The reading from all wells was subtracted with the reading from substrate control, which contains Red Substrate but no MMPs. Although different MMPs showed different cleavage rate on this substrate, the Red Substrate can detect the activity of sub-nanogram of all MMPs (n=3).
      NOTE: distinct purified MMP enzymes were used in this test. When using cell extracts, the kit will only detect a general MMP activity and it will not differentiate between the different MMPs.

    Protocols

    References for MMP Activity Assay Kit (Fluorometric - Red) (ab112147)

    ab112147 has not yet been referenced specifically in any publications.

    Product Wall

    We recommend starting with a 2 hour APMA activation step for a mixture of MMPs as in your smooth muscle cell extract.

    Ab112147 is for total MMP activity assay, though we do offer MMP3 detection kits as well. This kit will not differentiate between the different MMPs. On the graph displayed on the datasheet distinct purified MMP enzymes were used. The graph shows the ...

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    This kit uses a peptide linked to fluorescent probes providing a FRET signal. Once this peptide is cleaved the signal is quenched which provides the method of measuring the MMP activity. The peptide used is not specific for any one MMP and I can n...

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    As we discussed over the phone, I have checked into the specificity of the MMP Activity Assay Kit (ab112147). This kit uses a peptide linked to fluorescent probes providing a FRET signal. Once this peptide is cleaved the signal is quenched which p...

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    Thank you for your inquiry. Here are the answers to your questions.


    1. Why doesn't the kit include a positive control? --MMPs proteins are all very expensive. And since the kit can measure all of them and a customer might only be intere...

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    Thank you for your enquiry. I am sorry it has taken some time to obtain and clarify some information for you.

    I can confirm that ab112147 MMP Activity Assay Kit (Fluorometric - Red) is designed for measuring total MMP activity. The results s...

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    Thank you for contacting us.



    The MMPs are initially synthesized as inactive Zymogens with a pro-peptide domain that must be removed before the enzymeis active. Some MMPs have a prohormone convertase cleavage site as part of this ...

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    Thank you for your reply.

    Here are the answers to your questions.

    1 -Since I want to look at both MMP-1 and -13, I should use the 3hr incubation. At this time, MMP-1 will have been activated and MMP-13 should still be active. I will ...

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    Thank you for your reply.

    I have asked the lab about your enquiry and their response is below:

    The activation time for MMPs on the protocol are the suggested time that we find best for the specific MMP. For instance, 40 min incubation...

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    Thank you for calling Abcam earlier today.

    I have talked to the lab and they confirmed that the Ex wavelengths that you have are not compatible with this kit. The following kit would work for you though I believe (I would double check the Ex/E...

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    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"