Recombinant
RabMAb

Anti-MMP9 antibody [EP1254] (ab76003)

Overview

  • Product name
    Anti-MMP9 antibody [EP1254]
    See all MMP9 primary antibodies
  • Description
    Rabbit monoclonal [EP1254] to MMP9
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-P, Flow Cyt, ELISAmore details
  • Species reactivity
    Reacts with: Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human MMP9 aa 100-200.

  • Positive control
    • WB: U937, HL60 and TPA treated HT1080 cell lysates and rat kidney tissue lysate. IHC-P: Human gastic adenocarcinoma and spleen tissue. ICC/IF: HeLa, U-2 OS and domoic acid-treated U87-MG cells. Flow Cyt: A431 cells.
  • General notes

     

    This antibody works better in 1%SDS Hot Lysates in WB. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

    A trial size is available to purchase for this antibody.

    Mouse: We have internal testing data to indicate this antibody reacts with this species in immunohistochemical and ELISA-based applications, but we were unable to detect a band with mouse lysates in western blot. Please contact us for more information.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab76003 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
ICC/IF 1/250 - 1/500.
WB 1/1000 - 1/20000. Detects a band of approximately 92 kDa (predicted molecular weight: 78 kDa).
IHC-P 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

For unpurified use at 1/100 - 1/250.

Flow Cyt 1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ELISA Use at an assay dependent concentration.

Target

  • Function
    May play an essential role in local proteolysis of the extracellular matrix and in leukocyte migration. Could play a role in bone osteoclastic resorption. Cleaves KiSS1 at a Gly-
    -Leu bond. Cleaves type IV and type V collagen into large C-terminal three quarter fragments and shorter N-terminal one quarter fragments. Degrades fibronectin but not laminin or Pz-peptide.
  • Tissue specificity
    Produced by normal alveolar macrophages and granulocytes.
  • Involvement in disease
    Intervertebral disc disease
    Metaphyseal anadysplasia 2
  • Sequence similarities
    Belongs to the peptidase M10A family.
    Contains 3 fibronectin type-II domains.
    Contains 4 hemopexin repeats.
  • 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
    Processing of the precursor yields different active forms of 64, 67 and 82 kDa. Sequentially processing by MMP3 yields the 82 kDa matrix metalloproteinase-9.
    N- and O-glycosylated.
  • Cellular localization
    Secreted, extracellular space, extracellular matrix.
  • Information by UniProt
  • Database links
  • Alternative names
    • 82 kDa matrix metalloproteinase-9 antibody
    • 92 kDa gelatinase antibody
    • 92 kDa type IV collagenase antibody
    • CLG 4B antibody
    • CLG4B antibody
    • Collagenase Type 4 beta antibody
    • Collagenase type IV 92 KD antibody
    • EC 3.4.24.35 antibody
    • Gelatinase 92 KD antibody
    • Gelatinase B antibody
    • Gelatinase beta antibody
    • GelatinaseB antibody
    • GELB antibody
    • Macrophage gelatinase antibody
    • MANDP2 antibody
    • Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) antibody
    • Matrix Metalloproteinase 9 antibody
    • MMP 9 antibody
    • MMP-9 antibody
    • MMP9 antibody
    • MMP9_HUMAN antibody
    • Type V collagenase antibody
    see all

Images

  • All lanes : Anti-MMP9 antibody [EP1254] (ab76003) at 5 µg

    Lane 1 : Natural human MMP9 protein (dimer) (ab168863)
    Lane 2 : Natural human MMP9 protein (Proenzyme, monomer) (ab157344)
    Lane 3 : Recombinant Mouse MMP9 protein (ab39309)

    Lysates/proteins at 0.1 µg per lane.

    Secondary
    Infrared labelled goat anti-rabbit (green) antibody at 1/20000 dilution

    Performed under reducing conditions.

    Predicted band size : 78 kDa

    This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with anti-MMP9 antibody [EP1254] (ab76003; 5 microgram per mL)  overnight at 4°C. Antibody binding was detected using infrared labelled goat anti-rabbit  (green) antibody (diluted 1:20000) for 1 hour at room temperature before imaging.

  • Anti-MMP9 antibody [EP1254] (ab76003) at 1/1000 dilution (purified) + Rat kidney tissue lysate at 10 µg

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

    Predicted band size : 78 kDa
    Observed band size : 84-92 kDa (why is the actual band size different from the predicted?)

    Blocking and dilution buffer: 5% NFDM/TBST.

  • Immunocytochemistry/Immunofluorescence analysis of U-2 OS (human osteosarcoma) cells labelling MMP9 with ab76003 at 1/500 (4.3 μg/mL). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000, 2 μg/mL) was used as the secondary antibody. Cells were counter-stained with ab195889 Anti-Alpha Tubulin antibody [DM1A] (1/200, 2.5 μg/mL) - Microtubule Marker (Alexa Fluor® 594). DAPI (blue) was used as a nuclear counterstain.

    Confocal image showing cytoplasmic staining onU-2 OS cells, the expression increased after treatment with TPA (200 nM) for 24 hours (middle panel).

    Secondary Only Control: PBS was used instead of the primary antibody as the negative control with both TPA treated and untreated U-2 OS cells.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling MMP9 with purified ab76003 at 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • All lanes : Anti-MMP9 antibody [EP1254] (ab76003) at 1.5 µg/ml

    Lane 1 : Control U937 at 100 µg
    Lane 2 : Stimulated U937 (24 hours with 10 ng x mL-1 PMA (ab120297), 3 final hours with 3 ug x mL-1 of Brefeldin (ab120299)) at 100 µg
    Lane 3 : Human tonsils at 20 µg

    Secondary
    Goat anti-rabbit at 1/10000 dilution

    Predicted band size : 78 kDa
    Observed band size : 89 kDa (why is the actual band size different from the predicted?)

    Running buffer: MOPS.

    Conditions: Denatured/reduced.

    This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab76003 (rabbit-anti MMP9; 1.5 ug/mL) and ab8245 (loading control to GAPDH;  0.1 ug/mL) for 48 hours at 4°C. Before imaging, antibody binding was detected using infrared-labelled goat anti-rabbit (green) and goat anti-mouse (red) at  1:10,000 dilution for 1 hour at room temperature.

     

  • All lanes : Anti-MMP9 antibody [EP1254] (ab76003) at 1.5 µg/ml

    Lane 1 : Recombinant Human MMP9 protein (Proenzyme) (ab82955)
    Lane 2 : Recombinant Human MMP9 protein (Proenzyme) (ab82955)

    Lysates/proteins at 0.1 µg per lane.

    Secondary
    Goat anti-rabbit at 1/10000 dilution

    Predicted band size : 78 kDa
    Observed band size : 89 kDa (why is the actual band size different from the predicted?)

    Running buffer: MOPS.

    Conditions: Denatured/reduced.

    This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab76003 (rabbit-anti MMP9; 1.5 ug/mL) for 48 hours at 4°C. Before imaging, antibody binding was detected using infrared-labelled goat anti-rabbit (green) at  1:10,000 dilutions for 1 hour at room temperature.

  • Anti-MMP9 antibody [EP1254] (ab76003) at 1/2000 dilution (purified) + HT-1080 cell lysate at 10 µg

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

    Predicted band size : 78 kDa
    Observed band size : 84-92 kDa (why is the actual band size different from the predicted?)

    Blocking and dilution buffer: 5% NFDM/TBST.

  • Unpurified ab76003 staining MMP9 in U87-MG cells treated with domoic acid (ab120338), by ICC/IF. Increase of MMP9 expression correlates with increased concentration of domoic acid, as described in literature.
    The cells were incubated at 37°C for 6h in media containing different concentrations of ab120338 (domoic acid) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with unpurified ab76003 (1/200) dilution was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric adenocarcinoma tissue labelling MMP9 with unpurified ab76003 at a dilution of 1/100.

  • Overlay histogram showing permeabilized A431 cells stained with unpurified ab76003 (pink line). Negative control antibody (green line) was rabbit IgG.

  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD

References

This product has been referenced in:
  • Xu L  et al. Umbilical cord-derived mesenchymal stem cells on scaffolds facilitate collagen degradation via upregulation of MMP-9 in rat uterine scars. Stem Cell Res Ther 8:84 (2017). WB, IF, IHC ; Rat . Read more (PubMed: 28420433) »
  • Gong L  et al. Transthyretin regulates the migration and invasion of JEG-3 cells. Oncol Lett 13:1242-1246 (2017). WB ; Human . Read more (PubMed: 28454241) »

See all 87 Publications for this product

Customer reviews and Q&As

Filter by Application

Filter by Species

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Application
Western blot
Sample
Rat Tissue lysate - whole (kidney cortex and outer medulla)
Gel Running Conditions
Reduced Denaturing (10-20% Criterion SDS-HCl)
Loading amount
30 µg
Specification
kidney cortex and outer medulla
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Oct 27 2017

Application
Western blot
Sample
Mouse Tissue lysate - whole (Colon)
Gel Running Conditions
Reduced Denaturing (12)
Loading amount
25 µg
Treatment
7.6 mg AOM/kg body weight
Specification
Colon
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Username

Lewins Walter

Verified customer

Submitted Aug 03 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HTB94 chondrosarcoma)
Gel Running Conditions
Reduced Denaturing (7.5%)
Loading amount
10 µg
Specification
HTB94 chondrosarcoma
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 28 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (spleen)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate 6
Permeabilization
No
Specification
spleen
Blocking step
H2O2 as blocking agent for 10 minute(s) · Concentration: 3% · Temperature: RT°C
Fixative
formalin
Username

Abcam user community

Verified customer

Submitted Mar 30 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (HNSCC Tissue)
Antigen retrieval step
Heat mediated
Permeabilization
No
Specification
HNSCC Tissue
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde
Username

Dr. Biswanath Majumder

Verified customer

Submitted Jul 01 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Non-reduced Denaturing (7.5%)
Sample
Rat Tissue lysate - whole (ovaries)
Specification
ovaries
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Username

Abcam user community

Verified customer

Submitted Sep 30 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (MDA-MD-231)
Loading amount
25 µg
Specification
MDA-MD-231
Gel Running Conditions
Non-reduced Denaturing (8% PAGE SDS gel)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 02 2010

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

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