Overview

  • Product name
  • Description
    Rabbit polyclonal to MMP14
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Flow Cyt, IHC-P, IHC-Fr, WB, ICC, ICC/IF, IPmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to MMP14.

  • Positive control
    • Placenta, or Breast carcinoma.

Properties

Applications

Our Abpromise guarantee covers the use of ab3644 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
Flow Cyt Use at an assay dependent concentration. PubMed: 19910495

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

 

IHC-P 1/30.
IHC-Fr Use at an assay dependent concentration.
WB Use at an assay dependent concentration.
ICC Use a concentration of 1 µg/ml.
ICC/IF Use a concentration of 1 µg/ml.
IP Use at an assay dependent concentration. PubMed: 22002060

Target

  • Function
    Seems to specifically activate progelatinase A. May thus trigger invasion by tumor cells by activating progelatinase A on the tumor cell surface. May be involved in actin cytoskeleton reorganization by cleaving PTK7.
  • Tissue specificity
    Expressed in stromal cells of colon, breast, and head and neck. Expressed in lung tumors.
  • Sequence similarities
    Belongs to the peptidase M10A family.
    Contains 4 hemopexin-like domains.
  • Domain
    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
    modifications
    The precursor is cleaved by a furin endopeptidase.
  • Cellular localization
    Membrane. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt
  • Database links
  • Alternative names
    • Matrix metallopeptidase 14 (membrane inserted) antibody
    • Matrix metalloproteinase 14 antibody
    • Matrix metalloproteinase-14 antibody
    • Membrane type 1 matrix metalloproteinase antibody
    • Membrane type 1 metalloprotease antibody
    • Membrane type matrix metalloproteinase 1 antibody
    • Membrane-type matrix metalloproteinase 1 antibody
    • Membrane-type-1 matrix metalloproteinase antibody
    • MMP 14 antibody
    • MMP X1 antibody
    • MMP-14 antibody
    • MMP-X1 antibody
    • Mmp14 antibody
    • MMP14_HUMAN antibody
    • MMPX1 antibody
    • MT MMP 1 antibody
    • MT-MMP 1 antibody
    • MT1 MMP antibody
    • MT1-MMP antibody
    • MT1MMP antibody
    • MTMMP 1 antibody
    • MTMMP1 antibody
    see all

Images

  • All lanes : Anti-MMP14 antibody (ab3644) at 1/500 dilution

    Lane 1 : 15ug MCF-7 whole cell lysate
    Lane 2 : 15ug MSTO-2H11 whole cell lysate
    Lane 3 : 15ug Malme-3M whole cell lysate
    Lane 4 : 15ug Calu-1 whole cell lysate
    Lane 5 : 15ug Sk-Lu-1 whole cell lysate

    Secondary
    All lanes : Peroxidase-conjugated affinipure goat anti-rabbit

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 54,66 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 20 kDa (possible non-specific binding), 98 kDa (possible non-specific binding)


    Exposure time: 30 seconds


    This image is courtesy of an anonymous Abreview

    See Abreview

  • ICC/IF image of ab3644 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3644, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • IHC image of ab3644 staining in Hu Colon formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab3644, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

References

This product has been referenced in:
  • Neuhaus J  et al. Protease Expression Levels in Prostate Cancer Tissue Can Explain Prostate Cancer-Associated Seminal Biomarkers-An Explorative Concept Study. Int J Mol Sci 18:N/A (2017). Read more (PubMed: 28471417) »
  • Eiseler T  et al. Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion. J Biol Chem 291:462-77 (2016). Read more (PubMed: 26507660) »
See all 12 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.
I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx with ab51074.
To check the status of the order please contact our Customer Service team and reference this number.
Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.
I wish you the best of luck with your research.

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Answer

Following-up my message from Friday, I did receive more information regarding ab3644: the immunogen is a 12 amino acid peptide found between amino acids 100-200 of human MPP-14. Since the pro-peptide is aa 1-111, and the antibody recognizes the active as well as latent forms of the protein, we can narrow this range further to aa 112-200. I hope this helps. For ab16267, I am afraid there is no more information available.

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Answer

Thank you for your enquiry. I appreciate your continued patience in this matter. I have received some feedback from some of the sources of the antibodies that you are enquiring about; ab7033 - To follow on with the comments that I have made the source of mouse monoclonal [MMP2/2C1] to MMP2 (ab7033) was also concerned with regards the approach that you have used for your sample preparation. I appreciate that you have been using an NP40 buffer extraction. However, this is an approach designed for a cytoplasmic preparation of cell culture cells. I would like to follow up my previous email by recommending that you perform a loading control experiment using an antibody that targets a "housekeeping protein" for example GAPDH or beta actin. This can be performed under the conditions best recognized by the antibody you are using; most likely denaturing, reduced. This will enable you to fully determine the integrity of your protein with respect to its protein composition. My biggest concern with the blot images that you have provided me with are the band doublet that you have been detecting either side of the 37KDa marker as this is present in the majority of your blot images. The source of ab6586 - Collagen IV antibody (ab6586) makes a similar suggestion although Collagens should in fact be electrophoresed as you have been using non-denaturing, non-reduced conditions. However, it is important that the integrity of your samples are confirmed. I am still awaiting further information and am in the process of requesting blot images for the antibodies that you have enquired about. I appreciate your continued patience. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your enquiry. Once again I am sorry to hear of the difficulties that you have experienced using these antibodies. I have had a look through the information that you have provided in conjunction with the blot images and I have a few comments. Firstly I am concerned about the sample preparation that has been performed. Many of the extraneous bands that have been detected are significantly lower than I would expect. Curiously many are also doublets at approximately 25KDa and 37KDa. My suspicion would be non-specific labeling possibly due to protein degradation. You have highlighted that the samples were prepared as recommended by Abcam. Please can you detail exactly how this was performed as we make many recommendations for sample preparation. My concern with respect to the sample preparation is that many of the proteins that your are targeting are secreted proteins and therefore require delicate sample preparation. Can you tell me whether the skeletal muscle samples were postmortum, biopsies, surgical specimens etc. I would also appreciate details of the gel conditions that you are using. Similar to my previous response with respect to ab2500 I would appreciate it if you could provide me with details of the gel conditions. I would like to know whether these pre-cast gels are non-denaturing in addition to non-reduced. I would also like details of how the samples are prepared immediately prior to loading on the gel. Can you also provide me with details of the primary and secondary antibody dilutions that have been attempted for each of the samples. Given the non-specific reactivity that you have observed I am also interested whether any no-antibody control experiments have been performed to determine whether the extraneous bands are attricubatble to this reactivity. I additionally have a few comments with respect to individual antibodies that you have been applying: In the experiments that have been performed using ab1828, the secondary that has been employed is ab6721. This is an antibody that is specific for use by IHC and has not been tested for use in western blotting. We have received some excellent feedback as to the application of ab3644 through our Abreviews system. I would like to highlight the importance of a a suitable positive control. For MT1-MMP/MMP14 we recommend the use of placental or breast/lung tissue. For Collagen IV we recommend the use of human epidermal keratinocytes. I look forward to your comments on these matters.

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with these antibodies. The results that you have been obtaining are certainly troubling. What concerns me the most are the two bands that you are consistently detecting at approximately 25 and 37KDa. You are consistently detecing cross reacting bands and never detecting the endogenous target. Whilst some of the antibodies that you detail have not been tested using rat samples; potentially explaining the absence of reactivity with the endogenous band the presence of the two bands at the aforementioned molecular weight accross many of your samples seems to suggest problems with the secondary antibody. Especially in view of the fact that you have changed this antibody and are in fact using three independent secondary antibodies: Goat polyclonal to Rabbit IgG H&L (HRP) (ab6721) Goat polyclonal to Rabbit IgG + Mouse IgG & IgM - prediluted (HRP polymer) (ab2891) Rabbit polyclonal to Goat IgG H&L (HRP) (ab6741) I would appreciate it if you could provide me with protocol details of the approach that you are using so that I can better determine potential reasons for these reacting bands. We have not observed such reactivity with any of the antibodies you list. I am particularly interested in the generic protocol that you have been applying. To provide me with these details please click on the link below and complete our on line technical questionaire. If you have modified the protocol for any of the antibodies at any point please mention this. I am also very interested in the method of sample preparation and the control antibody (loading control) that you have used. https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=2500&mode=questionaire I look forward to hearing from you.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (cell lines: breast / lung)
Loading amount
15 µg
Specification
cell lines: breast / lung
Blocking step
Other as blocking agent for 30 minute(s) · Concentration: 0.05 % Twe

Abcam user community

Verified customer

Submitted May 04 2006

Answer

ab10800 -- Mouse monoclonal [HAS-3] to Integrin alpha 2 is provided at a concentration of 1mg/ml (this information was previosuly missing from the datasheet but has been added now). You do not mention which application the researcher plans to use the product, but knowing the concentration should now enable them to determine a starting dilution. There are also references on the datasheet that specifically feature the use of this antibody so please ask them to check here for further information. Positive controls: ab3644 -- Rabbit polyclonal to MMP14 Suitable tissue control sections for Immunohistochemistry: Placenta. Note the antibody cross reacts with human. This information is found on the positive control and cross reactivity sections of the datasheet. ab13018 -- Rabbit polyclonal to IL8 Receptor alpha There are beautiful IHC images on the datasheet showing staining of human skin, eccrine sweat glands. ab7747 -- Rabbit polyclonal to IL8 Shown to cross reacts with Human, Cynomolgus Monkey and Rhesus monkey. Product specific reference included on the datasheet is Fujii A et al. Differential expression of chemokines, chemokine receptors, cytokines and cytokine receptors in diffuse large B cell malignant lymphoma. Int J Oncol 24:529-38 (2004). PubMed: 14767537.

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