MMP Activity Assay Kit (Fluorometric - Green) (ab112146)

Overview

  • Product name
    MMP Activity Assay Kit (Fluorometric - Green)
    See all MMP kits
  • Detection method
    Fluorescent
  • Sample type
    Cell culture extracts, Purified protein
  • Assay type
    Direct
  • Product overview

    ab112146 MMP Activity Assay Kit uses a fluorescence resonance energy transfer (FRET) peptide as a generic MMP activity indicator. It is designed to check the general activity of an MMP enzyme and to screen MMP inhibitors. In the intact FRET peptide, the fluorescence of one part is quenched by another. After cleavage into two separate fragments by MMPs, the fluorescence is recovered. With excellent fluorescence quantum yield and longer wavelength, the probe is much more sensitive than an EDANS/Dabcyl FRET substrate. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/525 nm.

    Visit our FAQs page for tips and troubleshooting.


    The kit measures total MMP activity. It does not give an individual read-out for each MMP. The substrate peptide is a sequence that is recognized by all MMPs. A specific inihibitor would need to be included to determine the activity of a specific MMP enzyme.

  • Tested applications
    Suitable for: Functional Studiesmore details
  • Platform
    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab112146 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.

Images

  • Khajah et al investigates endocrine resistant breast cancer cells in response to changes in extracellular pH. MMP activity of pII cells was determined using ab112146. Cells were cultured in a gassed or ungasssed incubator for 1 hour and treated with EGF at 50ng/ml for 30 minutes. MMP activity was then determined using fluorogenic substrate. Fluorescence was measured for excitation/emission of 490/525 nm.

  • The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (6.25e4 cells) of cell lysate. Cells (1e6-5e7 cells) were sonicated in 0.3 mL RIPA buffer with protease inhibitors. Protein amount was determined by BCA method.

    Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).

    Purified MMPs used:

    MMP1 – ab134442

    MMP2 – ab125181

    MMP3 – ab96555

    MMP8 – ab168050

    MMP9 – ab157344

    MMP13 – ab134452

    MMP14 – ab157068

  • The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (6.25e4 cells) of cell lysate. Cells (1e6-5e7 cells) were sonicated in 0.3 mL RIPA buffer with protease inhibitors. Protein amount was determined by BCA method. Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).

    Purified MMPs used:

    MMP1 – ab134442

    MMP2 – ab125181

    MMP3 – ab96555

    MMP8 – ab168050

    MMP9 – ab157344

    MMP13 – ab134452

    MMP14 – ab157068

  • The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (0.56 mg) of mouse liver tissue lysate. Frozen tissue was homogenised in 1.2 ml RIPA buffer with protease inhibitors with 1 mm glass beads in a bead beater. Protein was determined by BCA method.

    Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).

     

    Purified MMPs used:

    MMP1 – ab134442

    MMP2 – ab125181

    MMP3 – ab96555

    MMP8 – ab168050

    MMP9 – ab157344

    MMP13 – ab134452

    MMP14 – ab157068

  • The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (0.21 mg) of mouse leg muscle tissue lysate. Frozen tissue was homogenised in 1.2 ml RIPA buffer with protease inhibitors with 1 mm glass beads in a bead beater. Protein was determined by BCA method. Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).

     

    Purified MMPs used:

    MMP1 – ab134442

    MMP2 – ab125181

    MMP3 – ab96555

    MMP8 – ab168050

    MMP9 – ab157344

    MMP13 – ab134452

    MMP14 – ab157068

  • The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (0.17 mg) of mouse heart tissue lysate. Frozen tissue was homogenised in 1.2 ml RIPA buffer with protease inhibitors with 1 mm glass beads in a bead beater. Protein was determined by BCA method. Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).

     

    Purified MMPs used:

    MMP1 – ab134442

    MMP2 – ab125181

    MMP3 – ab96555

    MMP8 – ab168050

    MMP9 – ab157344

    MMP13 – ab134452

    MMP14 – ab157068

  • The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (0.80 mg) of mouse brain tissue lysate. Frozen tissue was homogenised in 1.2 ml RIPA buffer with protease inhibitors with 1 mm glass beads in a bead beater. Protein was determined by BCA method. Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).

     

    Purified MMPs used:

    MMP1 – ab134442

    MMP2 – ab125181

    MMP3 – ab96555

    MMP8 – ab168050

    MMP9 – ab157344

    MMP13 – ab134452

    MMP14 – ab157068

     

  • Detection of activity of MMPs using ab112146.

    APMA-activated purified MMPs (30 ng each) were mixed with Green substrate. The fluorescence signal was monitored after 1 hour by using a microplate reader at Ex/Em = 490/525 nm. The reading from all wells was subtracted with the reading from substrate control. Although different MMPs showed different cleavage rate on this MMP substrate, the MMP Green 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

This product has been referenced in:
  • Incio J  et al. Metformin Reduces Desmoplasia in Pancreatic Cancer by Reprogramming Stellate Cells and Tumor-Associated Macrophages. PLoS One 10:e0141392 (2015). WB ; Mouse . Read more (PubMed: 26641266) »
  • Khajah MA  et al. Extracellular Alkaline pH Leads to Increased Metastatic Potential of Estrogen Receptor Silenced Endocrine Resistant Breast Cancer Cells. PLoS One 8:e76327 (2013). Functional Studies . Read more (PubMed: 24098477) »

See all 3 Publications for this product

Customer reviews and Q&As

For ab112146, you are only measuring general MMP activity, so although there are guidelines about which time points represent which MMPs, you cannot correlate the times to each MMP or discern each one individually. In other words, you cannot take a rea...

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good for a quick look

Good Excellent 5/5 (Ease of Use)
Abreviews
pros:
-Very easy to use
-Good way to look at overall MMP activity.

cons:
-Instructions should be clarified (it almost looks like it was google-translated)
-No standards
-No plates
-Enzyme activation time is different depending on what MMPs you're interested in. Can be a little confusing.
Username

Abcam user community

Verified customer

Submitted Nov 07 2016

Comme indiqué sur la fiche technique d’ab112146, nous avons testé ce kit sur plusieurs tissus de souris : le foie, le muscle de jambe, le cœur et le cerveau.
Vous pouvez donc utiliser ce kit sur les protéines totales de l’aorte de souris.
En ...

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MMP Activity Assay Kit for Organ Homogenates

Average Good 4/5 (Ease of Use)
Abreviews
We tested this Kit for MMP Activity in Organhomogenates (liver and spleen).
The Organhomogenates were prepared with RIPA-Buffer.
We used 10µg total protein for this assay.
The Assay was used as indicated in the protocol booklet.
For our test approach, we did not know, what MMP could be active in our homogenates. Since the primary activation step for the MMPs is MMP-specific, we had to select the incubation time for the MMPs rather than could analyze pan-MMP activation.
In our experimental setup, we did not detect specific MMP activation, since no appropriate positive control was provided within this kit.
In conclusion, technically, the assay can be performed with organ homogenates, but it is necessary to know at which MMP you want to analyze and in optimal cases perform a positive control with spiked MMP-protein in your organ homogenates
Username

Abcam user community

Verified customer

Submitted Jan 09 2014

Thank you for your enquiry.

I am pleased to help answer your questions:

1. Does is pick up MMP activity from both human and mouse samples? Has it been tested in these species and is there any data?

Only recombinant protein...

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Thank you for contacting us.

You will have to do a MMP standard curve to get EC50, and usually use a EC80 of the enzyme to do your inhibitor study to get the inhibition study to get IC50.

Hope it helps. Thanks!

After our phone conversation, I am also emailing you the information for your records. I am sorry about the confusion and we will update our protocol shortly.

1) How much volume of 1mM APMA solution shouldbe added to the samples?
Answer...

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Thanks for your inquiry.  I have contacted the Lab and they have confirmed that purified MMPs was used for the assay. It is most likely the samples don't have enough activated MMPs.  Please make sure that you have a real positive control (purifie...

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Thank you for your recent phone call. This will be a special order for you. The catalogue number will be ab118834, MMP Green™ reference standard, 1 mM in DMSO, 100 uL. The new order number is: XXXXXX. We are expecting to get this item from the US o...

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Thank you for your patience. We can send you the DMSO stock solution substrate as a free sample for you. We are waiting for this item from our US office and will ship it to you as soon as possible. Could you please get back to me and co...

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

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