Anti-MMP9 抗体 [EP1254]

• WB

Lab

Western blot - Anti-MMP9 antibody [EP1254] (AB76003)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Exposure time : 3 seconds; 40 seconds. ab181602 was used as loading control for GAPDH. Although MMP9 has been studied in brain in some publications, ab76003 was unable to detect signal in normal brain tissue， this may because MMP9 expression level is low in normal brain and would be increased in abnormal conditions like injury (PMID : 31198417).

All lanes:

Western blot - Anti-MMP9 antibody [EP1254] (ab76003) at 1/1000 dilution

Lane 1:

Human lung tissue lysate at 20 µg

Lane 2:

Human brain tissue lysate at 20 µg

Lane 3:

Human spleen tissue lysate at 20 µg

Lane 4:

Human lymph node tissue lysate at 20 µg

Lane 5:

Rat lung tissue lysate at 20 µg

Lane 6:

Rat brain tissue lysate at 20 µg

Lane 7:

Rat spleen tissue lysate at 20 µg

Lane 8:

Rat kidney tissue lysate at 20 µg

Lane 9:

Rat lymph node tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 29 kDa

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• Functional Study

Unknown

Functional Study - Anti-MMP9 antibody [EP1254] (AB76003)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [EP1254] (AB76003)

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

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MMP9 antibody [EP1254] (AB76003)

Overlay histogram showing permeabilized A431 (Human epidermoid carcinoma cell line) cells stained with unpurified ab76003 (pink line).

Negative control antibody (green line) was rabbit IgG.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [EP1254] (AB76003)

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

Negative control using PBS instead of primary antibody (inset).

Counterstained with hematoxylin.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MMP9 antibody [EP1254] (AB76003)

Immunocytochemistry/Immunofluorescence analysis of U-2 OS (human osteosarcoma) cells labeling 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 counterstained 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.

• WB

Lab

Western blot - Anti-MMP9 antibody [EP1254] (AB76003)

Western blot : Rabbit Monoclonal[EP1254] to MMP9 76003 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 90 kDa in Wild-type A549 TPA-treated (80nM, 24h) cell lysates with no signal observed at this size in MMP9 knockout A549 TPA-treated (80nM, 24h) cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-MMP9 antibody [EP1254] (ab76003) at 1/1000 dilution

Lane 1:

Wild-type A549 TPA-treated (80nM, 24h) at 20 µg

Lane 2:

Wild-type A549 Vehicle Control TPA (0nM, 24h) at 20 µg

Lanes 3 - 4:

Western blot - Human MMP9 knockout A549 cell line (ab286527) at 20 µg

Lane 5:

Human Lung at 20 µg

Lane 6:

MOLT-4 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 78 kDa

Observed band size: 90 kDa

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• WB

AbReview55690****

Western blot - Anti-MMP9 antibody [EP1254] (AB76003)

All lanes:

Western blot - Anti-MMP9 antibody [EP1254] (ab76003) at 1/5000 dilution

Lanes 1 - 2:

HTB94 (human chondrosarcoma cell line) cell lysate at 10 µg

Lanes 3 - 4:

HTB94 (human chondrosarcoma cell line) conditioned media at 10 µg

Secondary

All lanes:

Goat anti-rabbit IgG at 1/5000 dilution

Predicted band size: 78 kDa

true

Data courtesy of an anonymous AbReview

• WB

Lab

Western blot - Anti-MMP9 antibody [EP1254] (AB76003)

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-labeled goat anti-rabbit (green) and goat anti-mouse (red) at 1 : 10,000 dilution for 1 hour at room temperature.

All lanes:

Western blot - 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 (<a href='/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>), 3 final hours with 3 ug x mL-1 of Brefeldin (<a href='/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)) at 100 µg

Lane 3:

Human tonsils at 20 µg

Secondary

All lanes:

Goat anti-rabbit at 1/10000 dilution

Predicted band size: 78 kDa

Observed band size: 89 kDa

false

• WB

Supplier Data

Western blot - Anti-MMP9 antibody [EP1254] (AB76003)

Blocking and dilution buffer : 5% NFDM/TBST.

The expression of MMP-9 can be stimulated by various agents, such as inflammatory cytokine, growth factor, and 12-O-tetradecanoylphorbol-13-acetate (TPA) (PMID : 21047770, 28969043).

Based on our preliminary data, ab76003 detects no or weak band of interest in the untreated cell lines at the dilution of 1/200. Treatment increasing the expression of MMP-9 is recommended when using this antibody.

All lanes:

Western blot - Anti-MMP9 antibody [EP1254] (ab76003) at 1/200 dilution

Lane 1:

LoVo (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Huh7 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

A549 (Human lung carcinoma epithelial cell) serum starved overnight whole cell lysate at 20 µg

Lane 7:

A549 (Human lung carcinoma epithelial cell) serum starved overnight, then treated with 80nM TPA for 24 hours whole cell lysate at 20 µg

Lane 8:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) serum starved overnight whole cell lysate at 20 µg

Lane 9:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) serum starved overnight, then treated with 200nM TPA for 24 hours whole cell lysate at 20 µg

Lane 10:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 78 kDa

Observed band size: 84-82 kDa

false

Exposure time: 60s

• WB

Lab

Western blot - Anti-MMP9 antibody [EP1254] (AB76003)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MMP9 antibody [EP1254] (ab76003) at 1/1000 dilution

All lanes:

Rat kidney tissue lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 78 kDa

Observed band size: 84-92 kDa

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• WB

Lab

Western blot - Anti-MMP9 antibody [EP1254] (AB76003)

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 labeled goat anti-rabbit (green) antibody (diluted 1 : 20000) for 1 hour at room temperature before imaging.

Lanes 1 - 2:

Western blot - Anti-MMP9 antibody [EP1254] (ab76003) at 5 µg

Lane 3:

Western blot - Anti-MMP9 antibody [EP1254] (ab76003) at 5 µg/mL

Lane 1:

Western blot - Native human MMP9 protein (dimer) (<a href='/products/proteins-peptides/native-human-mmp9-protein-dimer-ab168863'>ab168863</a>) at 0.1 µg

Lane 2:

Western blot - Native human MMP9 protein (Proenzyme, monomer) (<a href='/products/proteins-peptides/native-human-mmp9-protein-proenzyme-monomer-ab157344'>ab157344</a>) at 0.1 µg

Lane 3:

Western blot - Recombinant Mouse MMP9 protein (<a href='/products/proteins-peptides/recombinant-mouse-mmp9-protein-ab39309'>ab39309</a>) at 0.1 µg

Secondary

All lanes:

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

Predicted band size: 78 kDa

false

• WB

Unknown

Western blot - Anti-MMP9 antibody [EP1254] (AB76003)

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-labeled goat anti-rabbit (green) at 1 : 10,000 dilutions for 1 hour at room temperature.

All lanes:

Western blot - Anti-MMP9 antibody [EP1254] (ab76003) at 1.5 µg/mL

All lanes:

Western blot - Recombinant Human MMP9 protein (Proenzyme) (<a href='/products/proteins-peptides/recombinant-human-mmp9-protein-proenzyme-ab82955'>ab82955</a>) at 0.1 µg

Secondary

All lanes:

Goat anti-rabbit at 1/10000 dilution

Predicted band size: 78 kDa

Observed band size: 89 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-MMP9 antibody [EP1254] (AB76003)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.