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MMP Activity Assay试剂盒(Fluorometric - Green) (ab112146)

Reviews (6)Q&A (8)References (48)
Functional studies - MMP Activity assay kit (Flurometric - Green) (ab112146)
  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)

Key features and details

  • Detection method: Fluorescent
  • Platform: Microplate reader
  • Assay time: 60 min
  • Sample type: Cell culture supernatant, Purified protein

概述

  • 产品名称

    MMP Activity Assay试剂盒(Fluorometric - Green)
    参阅全部 MMP 试剂盒

  • 检测方法

    Fluorescent

  • 样品类型

    Cell culture supernatant, Purified protein

  • 检测时间

    0h 60m

  • 产品概述

    MMP Activity Assay Kit ab112146 is designed to check the general activity of an MMP enzyme and to screen MMP inhibitors.


    The MMP assay protocol uses a fluorescence resonance energy transfer (FRET) peptide as a generic MMP activity indicator. 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. The probe signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/525 nm.


    MMP activity assay protocol summary:
    - activate pro-MMPs with APMA for between 20 min and 24 hrs (depending on which MMP is of interest)
    - add samples to wells
    - add MMP Green substrate
    - analyze with a fluorescent microplate reader, either after 30-60 mins for end-point reading, or every 5 min for 30-60 min for kinetic reading

  • 说明

    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.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

  • 平台

    Microplate reader

性能

图片

  • Functional studies - MMP Activity assay kit (Flurometric - Green) (ab112146)
    Functional studies - MMP Activity assay kit (Flurometric - Green) (ab112146)Khajah MA et al., PLoS One 8(10), Fig 8e. doi: 10.1371/journal.pone.0076327. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    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.

  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
    Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)

    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

  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
    Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)

    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

  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
    Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)

    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

  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
    Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)

    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

  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
    Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)

    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

  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
    Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)

    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

     

  • Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
    Functional Studies - MMP Activity Assay Kit (Fluorometric - Green) (ab112146)
    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.

文献 (48)

发表研究结果有使用 ab112146？请让我们知道，以便我们可以引用本数据表中的参考文章。

ab112146 被引用在 48 文献中.

  • Jung J  et al. Cysteamine Suppresses Cancer Cell Invasion and Migration in Glioblastoma through Inhibition of Matrix Metalloproteinase Activity. Cancers (Basel) 16:N/A (2024). PubMed: 38893149
  • Obi AT  et al. Experimental venous thrombus resolution is driven by IL-6 mediated monocyte actions. Sci Rep 13:3253 (2023). PubMed: 36828892
  • Wu CH  et al. Zinc Finger Protein 90 Knockdown Promotes Cisplatin Sensitivity via Nrf2/HO-1 Pathway in Ovarian Cancer Cell. Cancers (Basel) 15:N/A (2023). PubMed: 36900383
  • Mousavizadeh R  et al. Exposure to oxLDL impairs TGF-β activity in human tendon cells. BMC Musculoskelet Disord 24:197 (2023). PubMed: 36927534
  • Vadovics M  et al. Candida albicans Enhances the Progression of Oral Squamous Cell Carcinoma In Vitro and In Vivo. mBio 13:e0314421 (2022). PubMed: 35089096
View all Publications for this product

客户评价及客户问答

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1-10 of 14 Abreviews or Q&A

General MMP activity in cell lysate of MCF-7 cells

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
The MMP Activity Assay Kit (ab112146) was applied to determine total MMP activity in the cell lysate of MCF-7 cells. In our experimental setup, we detected the general MMP activation in MCF-7 breast cancer cells towards the selected compound and the known MMP inhibitor Ilomastat (ab120845) as a reference. We strictly followed the provided protocol. It is an easy and helpful experiment to know the overall MMP activity. But no known inhibitor is provided in this kit.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Feb 18 2021

Easy and quick assay

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
Very quick and easy detection kit. Worked very well
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Nov 30 2020

MMP activity in Caco-2 supernatant stimulated with E. coli or MMP inhibitor BB-94

 Below average Excellent 5/5 (Ease of Use)
Abreviews
abreview image
We stimulated Caco-2 cells with E. coli (O6:Hnt) at MOI 5 for 4 h in pre and absence of MMP inhibitor BB-94. We collected supernatant of Caco-2 cells and measured MMP activity using ab112146. Diagram shows the result of one experiment with technical replicates.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Feb 15 2019

MMP activity in MSC culture supernatants

 Good Good 4/5 (Ease of Use)
Abreviews
abreview image
The MMP Activity Assay Kit was used to determine MMP Activity in supernatants from human mesenchymal stromal cell culture on different substrates. Supernatant samples were collected after 2 days of cell culture, stored at -80°C and thawed on ice prior to the analysis.
The assay working solutions were prepared: APMA working solution – component B diluted 1:500 in assay buffer – and MMP Green Substrate working solution – MMP Green Substrate (fluorescent dye) diluted 1:100 in assay buffer.
In a 384 well plate, 10 µL of each sample was pipetted in duplicates. One reference sample of solely assay buffer (40 µL), one with assay buffer (30 µL) and component B working solution (10 µL) and one reference with assay buffer (10 µL), APMA working solution (10 µL) and MMP Green Substrate working solution (20 µL) were also prepared in duplicates – the working solutions were added at the same time as for the samples from the experiments.
APMA working solution was added in 1:1 ratio (10 µL) to each sample on ice. To activate the MMPs for measuring, specifically MMP1 (Collagenase) and MMP14 (MT-MMP) based on the incubation time chosen, the samples were incubated at 37°C for 3 h. Then, fluorescent dye – MMP Green Substrate working solution – was added in a 1:2 ratio to the sample volume (20 µL) on ice. The samples were incubated at RT on a gentle shaker for 30 min and the fluorescence was measured at Ex/Em 490/525 nm.
The data shown were obtained from 4 independent replicates.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Sep 10 2018

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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Nov 07 2016

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
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Jan 09 2014

Question


 
Is this product capable of distinguishing between the activities of different MMPs? For example, let's say that my biologic samples has both MMP9 and MMP13. According to the Appendix in the protocol book, the incubation times of my samples with the APMA should be 40min for MMP13 and 2h for MMP9. So if I took a reading at 40min, then fluorescence intensity corresponds to the activity of MMP13. But does the fluorescence reading taken at 2h represent the cumulative activity of both MMP9 and MMP13 (and any other unknown MMPs that may be contributing to the signal)??

Read More
Answer

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 reading at 45 min and say it is MMP13 activity and take a reading at 2h and say it is MMP9 activity. The kit is for general activity only.

Read More

Question

I would like to know if i can perform the MMP activity assay (ab112146) on total protein of mice aorta. Extraction was performed with an extraction kit from MM (Merck Millipore ref 2140 : http://www.millipore.com/catalogue/item/2140).

If yes, could you please indicate to me which amount of protein I’m supposed to use per sample.

Read More
Answer

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 ce qui concerne le taux de protéine à utiliser, car tous les échantillons sont différent, il faudrait faire un test préliminaire avec vos échantillons et tester plusieurs concentrations pour voir laquelle donne de bons résultats.
Dans nos tests faits au laboratoire, la quantité de protéine totale initiale dans les échantillons de tissus de souris utilisées était:

Muscle de la jambe 8.32 mg / ml
Foie 22,54 mg / ml
Cerveau 32,12 mg / ml
Coeur de 6,76 mg / ml
Un puits été composé de 50 ul d'échantillon de tissu de la souris et de 50 ul d'échantillon de MMP (tous avec la concentration de 0,0125 mg/ml).

Read More

Question

Hi Abcam tech,

I have received the following questions from a customer regarding the MMP Activity Assay Kit (Fluorometric - Green) (ab112146)

1. How long from the time of delivery or from the time the kit is opened does it expire?

2. Does is pick up MMP activity from both human and mouse samples?

3. We want to detect secreted MMP activity. Can culture supernatant be used in this assay? Alternatively, if we were to run the assay on adherent cells, how would we go about doing that?

Read More
Answer

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 proteins have been tested, it should be OK for both human and mouse samples theoretically.

2. We want to detect secreted MMP activity. Can culture supernatant be used in this assay?

It should be fine to use culture supernatant. We woudl advise to use phenol red free medium as this can interfere with the assay.

3. Alternatively, if we were to run the assay on adherent cells, how would we go about doing that?

We would suggest to try a traditional freeze-thawed method

I hope this information is helpful. Should you have any questions, please do not hesitate to contact us.

Read More

Question

Under the data analysis section it says you can get
inhibition %, EC50, IC50, etc data, how do I anbalyze the data to get these numbers?

Read More
Answer

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!

Read More

1-10 of 14 Abreviews or Q&A

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