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

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
  • 测试
    1 x 100 test
  • 样品类型
    Cell culture extracts, Adherent cells, Suspension cells, Purified protein
  • 检测类型Direct
  • 产品概述

    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.

  • 经测试应用适用于: Functional Studiesmore details
  • 平台Microplate

性能

    应用

    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.

    应用 Ab评论 说明
    Functional Studies Use at an assay dependent concentration.

    MMP Activity Assay Kit (Fluorometric - Green) 图像

    • 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.

    实验方案

    MMP Activity Assay Kit (Fluorometric - Green) (ab112146)参考文献

    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

    Product Wall

    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

    提交于 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 ...

    Read More

    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

    提交于 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...

    Read More

    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...

    Read More

    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...

    Read More

    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...

    Read More

    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...

    Read More

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