Anti-MMP9 antibody (ab38898)

Rabbit polyclonal MMP9 antibody. Validated in WB, IP, ELISA, IHC, ICC, ICC/IF and tested in Mouse, Rat, Dog, Human. Cited in 299 publication(s). Independently reviewed in 44 review(s).

Overview

  • Product name
  • Description
    Rabbit polyclonal to MMP9
  • Host species
    Rabbit
  • Specificity
    The antibody binds to Gelatinase-B, but does not cross react with the other MMP family members (MMP-1, MMP-2, MMP-3). In our hands, we observe a weaker signal in WB in human samples compared to mouse samples (BLAST of full length mouse protein sequence showed 72% homology with the Human MMP9 sequence).
  • Tested applications
    Suitable for: IHC-P, IHC-Fr, WB, IP, ELISA, ICC/IF, ICC, IHC-FoFrmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Dog, Human
  • Immunogen

    Full length protein corresponding to Mouse MMP9.

  • Positive control
    • IHC-Fr: Mouse liver and heart tissue. IHC-P: Mouse lung and pancreatic carcinoma tissue; Zebrafish pancreas tissue WB: Recombinant Human MMP9 protein; U937 and Raw 264.7 cell lysate. ICC/IF: Mouse neutrophils and monocytes.
  • General notes

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this antibody.

Applications

Our Abpromise guarantee covers the use of ab38898 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
IHC-P 1/100 - 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-Fr 1/1000.

(see Abreview submitted by Greg Gibson)

We recommend using Goat Anti-Rabbit IgG H&L (Cy3 ®) preadsorbed (ab6939) secondary antibody

WB 1/1000. Detects a band of approximately 92 kDa. When using colorimetric substrates such as BCIP/NBT use at a 1/5000 dilution (for chemiluminescent substrates). Detects a band of approximately 92-95 kDa (pro-form) and 82kDa (active form) (Human samples). Mouse MMP9 is larger, and on SDS PAGE gels runs about 102-105 kDa. Dilution optimised using Chromogenic detection.
IP Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
ICC/IF 1/500.
ICC Use at an assay dependent concentration.
IHC-FoFr Use at an assay dependent concentration. PubMed: 19295156

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

  • Immunofluorescence staining showing MMP9 (green) and DAPI (blue) in 12 weeks old N-IF and 24αβNOD control mouse livers. Scale bars are 100 μm.

    Immunohistochemical staining was performed on liver biopsies fixed in 4% paraformaldehyde and embedded in OCT. The frozen tissues were cut in 5 μm thick sections and stained using primary antibody against matrix metallopeptidase 9 (MMP9) (1:300, Abcam, ab38898). The nuclei were visualized with DAPI. The sections where analyzed using confocal microscopy.

  • MMP-9 immunohistochemistry in the lung tissue of a C3HeB/FeJ mouse, 10 weeks after M. tuberculosis aerosol infection. MMP-9 was visualized in macrophages (black solid arrows) and neutrophils (black dotted arrows).

    Paraffin embedded sections were rehydrated in graded alcohols, steamed in citrate buffer at pH 6 and probed at room temperature for 2 hours using the MMP-9 (rabbit polyclonal; 1:250; Abcam [AB38898]) and processed with a polymer-HRP kit (BioGenex) with diaminobenzidine development and Mayer hematoxylin counterstaining. Lungs from uninfected, and infected but untreated animals without primary antibody served as negative controls. Slides were scanned using the Apeiro digital scanner (Leica).

  • All lanes : Anti-MMP9 antibody (ab38898) at 5 µg/ml

    Lane 1 : Recombinant Human MMP9, His tagged (ab82955) at 0.1 µg
    Lane 2 : U937 whole cell lysate at 100 µg
    Lane 3 : U937 whole cell lysate - treated with PMA and Brefeldin (24 hour treatment) at 100 µg
    Lane 4 : Raw 264.7 (Mouse) whole cell lysate at 100 µg
    Lane 5 : Raw 264.7 (Mouse) whole cell lysate - treated with LPS (6 hour treatment, 1ug/mL) at 100 µg

    Performed under reducing conditions.


    ab38898 detects recombinant Human MMP9 running at ~85 kDa, and endogenous full-length MMP9 in LPS-stimulated cells at ~100 kDa. This antibody also detects a band at 90 kDa in U937 PMA-treated cells. 

     

    ab38898 was incubated at 5 ug/mL and ab8245 (loading control to GAPDH) was diluted at 0.1 ug/mL and both were incubated for 48 hours at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

  • ab38898 at a 1/1000 dilution staining mouse heart tissue by Immunohistochemistry (Frozen sections). The tissue was removed from a mouse, rinsed in PBS and slowly frozen in supercooled isopentane. 14um sections were made using a cryostat. The sections were acetone fixed and blocked in 2% BSA prior to incubation with the MMP9 antibody. Goat Anti-Rabbit IgG H&L (Cy3 ®) preadsorbed (ab6939)  was used as the secondary antibody. In the image: red staining = MMP9, blue staining = nuclei, green = gelatinase activity.

  • ab38898 staining MMP9 in 6 month-old transgenic zebrafish pancreas (Ihha overexpression) by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

    Sections were incubated with primary antibody (1/500) and HRP-conjugated secondary antibody colored using DAB solution. Slides were counterstained with hematoxylin.
  • All lanes : Anti-MMP9 antibody (ab38898) at 2 µg/ml

    Lane 1 : Native human MMP9 protein (Proenzyme, monomer) (ab157344)
    Lane 2 : Recombinant Mouse MMP9 protein (ab39309)

    Lysates/proteins at 0.1 µg per lane.

    Secondary
    All lanes : Infrared labelled goat anti-rabbit (green) at 1/20000 dilution

    Performed under reducing conditions.


    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 (ab38898; 2 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.

  • ab38898 staining MMP9 (red) in Mouse Neutrophils and Monocytes cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 2%BSA + 0.2% tritonX100 in PBS. Samples were incubated with primary antibody (1/200 in 2%BSA + 0.2% tritonX100 in PBS) for 25 minutes at 23°C. An Alexa Fluor® 568-conjugated Donkey anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody. DAPI is stained blue

    See Abreview

  • Anti-MMP9 antibody (ab38898) + Human MMP9

    Observed band size: 88,92 kDa
    why is the actual band size different from the predicted?



    Ab38898 detects a band at 92 Kd (pro-form) and a band at 88 Kd (active form). Mouse MMP9 is slightly larger than human MMP9, and the antibody detects a band at about 105 Kd. It is recommend to concentrate samples by Gelatin-agarose affinity chromatography prior to Western blot usage. A recommended starting concentration for Western blots is 1:1000 when using colorimetric substrates such as BCIP/NBT, and 1:5000 for chemiluminescent substrates. Higher concentration of antibody may be needed for non-human samples.
  • ab38898 staining MMP9 in Mouse Pancreatic carcinoma tissue sections by IHC-P (formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 1 hour at room temperature. Antigen retrieval was by heat mediation in citric acid (pH6). Samples were incubated with primary antibody (1/100) in 1% Aurion BSA for 12 hours. An HRP-conjugated Donkey polyclonal to rabbit IgG (1/100) was used as secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Xue F  et al. Neferine inhibits growth and migration of gastrointestinal stromal tumor cell line GIST-T1 by up-regulation of miR-449a. Biomed Pharmacother 109:1951-1959 (2019). Read more (PubMed: 30551450) »
  • Liu X  et al. Immunohistochemical analysis of matrix metalloproteinase-9 predicts papillary thyroid carcinoma prognosis. Oncol Lett 17:2308-2316 (2019). Read more (PubMed: 30675296) »
See all 308 Publications for this product

Customer reviews and Q&As

1-3 of 3 Q&A

Answer

Product Anti-MMP9 antibody (ab38898)is specific to MMP-9, and will not cross-react with fibronectin..

However, please note that MMP-9 binds to fibronectin, and fibronectin co-elutes with MMP-9 in affinity chromatography procedures. In plasma and tissue culture media samples, MMP-9 can be found associated with fibronectin.

We have observed a complex of MMP-9 and fibronectin in SDS-PAGE applications, and it can be surprisingly difficult to dissociate the complex. Therefore, it is possible that product ab38898 is able to detect a complex of MMP-9 and fibronectin in certain applications; however, ab38898 is specific to MMP-9 alone.

Read More

Answer

Product Anti-MMP9 antibody (ab38898) is a polyclonal Ab raised against the full length mouse MMP-9 sequence, and will detect both the pro-form and the active form of the MMP-9 protein.

Read More

Answer

Product Anti-MMP9 antibody (ab38898) is a polyclonal Ab generated against full length mouse MMP-9; this Ab will detect both the pro-form and active form of mouse and human MMP-9.

Mouse MMP-9 is slightly larger than human MMP-9. Human MMP-9 has a calculated MW of approximately 78.5 kDa. However, human MMP-9 is glycosylated at several amino acid residues and due to this glycosylation runs at an observed MW of approximately 92 kDa on non-reducing SDS-PAGE gels, and an observed MW of approximately 95 kDa on reducing gels.

Mouse MMP-9 has a calculated MW of approximately 80.5 kDa, and is more heavily glycosylated than its human ortholog. Mouse MMP-9 therefore runs at an observed MW of approximately 105 kDa on reducing SDS-PAGE gels.

In both mouse and human, MMP-9 is cleaved into a variety of forms: first by loss of the signal sequence, then by the loss of the propeptide domain, followed by a succession of cleavage events at the carboxyterminal hemopexin-like domain. These cleavage events can generate an apparent MW ladder of MMP-9 forms, running from the latent enzyme (approximately 100 kDa) to the smallest cleaved fragments in the 30 kDa range.

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