Anti-MMP9 antibody [RM1020] (ab283575)

Anti-MMP9 antibody [RM1020] (ab283575) is a rabbit recombinant multiclonal antibody that is used to detect MMP9 in Western Blot, Flow Cytometry (Intra), Flow Cytometry, IP, IHC-P, IHC-Fr, ICC/IF, ELISA. Suitable for Human, Mouse, Rat samples.

- Specificity confirmed with MMP9 knockout cell line validation

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Images

Western blot - Anti-MMP9 antibody [RM1020] (AB283575), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Multiclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

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Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	ICC/IF	IP	Flow Cyt (Intra)	WB	I-ELISA	IHC-Fr	IHC-P
Human	Tested	Expected	Tested	Tested	Expected	Expected	Tested
Mouse	Tested	Tested	Tested	Tested	Expected	Tested	Tested
Rat	Expected	Expected	Expected	Tested	Expected	Tested	Tested
Recombinant fragment - Mouse	Not recommended	Not recommended	Not recommended	Not recommended	Tested	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/50	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/50	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Mouse	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/30	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Human, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Mouse	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/500	Notes -
Species Human	Dilution info 1/500	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Mouse	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/1000	Notes -
Species Rat	Dilution info 1/1000	Notes -
Species Human	Dilution info 1/1000	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Mouse	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Recombinant fragment - Mouse	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Human, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/100	Notes -
Species Rat	Dilution info 1/100	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Human	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Mouse	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/5000	Notes -
Species Rat	Dilution info 1/5000	Notes -
Species Human	Dilution info 1/5000	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Mouse	Dilution info -	Notes -

Target data

See full target information MMP9

Function

The protein expressed by gene MMP9 is a matrix metalloproteinase that plays an essential role in the local proteolysis of the extracellular matrix and in leukocyte migration. It cleaves KiSS1 at a Gly-|-Leu bond and processes NINJ1 to generate the Secreted ninjurin-1 form. Additionally, MMP9 cleaves type IV and type V collagen into large C-terminal three-quarter fragments and shorter N-terminal one-quarter fragments. It degrades fibronectin but not laminin or Pz-peptide. This supplementary information is collated from multiple sources and compiled automatically.

Additional Targets

Mmp9

Alternative names

CLG4B, MMP9, Matrix metalloproteinase-9, MMP-9, 92 kDa gelatinase, 92 kDa type IV collagenase, Gelatinase B, GELB

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Multiclonal
Immunogens: The exact immunogen used to generate this antibody is proprietary information.
Clone number: RM1020
Purification technique: Affinity purification Protein A
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

What is this antibody validated in?

Anti-MMP9 antibody [RM1020] (ab283575) is a rabbit recombinant recombinant multiclonal antibody and is validated for use in Western Blot (WB), Flow Cytometry (Intra), Flow Cytometry (Flow Cyt), Immunoprecipitation (IP), Immunohistochemistry (IHC-P), Immunohistochemistry (IHC-Fr), Immunocytochemistry/immunofluorescence (ICC/IF), ELISA, in Human, Mouse, Rat samples.

What is the molecular weight of MMP9?

Anti-MMP9 [RM1020] (ab283575) specifically detects a band for MMP9 (UniProt: P14780) at a molecular weight of 78kDa.

Recommended positive controls

WB: U-2 OS treated with TPA , RAW 264.7 treated with LPS and BFA, NR8383, NR8383 treated with LPS and BFA, and Human lung and mouse lung lysates.IHC-P: Human spleen, Human tonsil, Mouse spleen and Rat spleen tissues.IHC-Fr: Mouse lung and Rat lung tissues.ICC/IF: RAW 264.7 cells treated with LPS and BFA, U-2 OS cells treated with TPA .Flow Cyt (intra): U-2 OS cells treated with TPA, RAW 264.7 cells treated with LPS and BFA.IP: Mouse lung lysate

Trusted by the scientific community

Anti-MMP9 [RM1020] (ab283575) was first used in a scientific publication in 2021 and has been cited over 30 times in peer-reviewed journals.

Trial sizes available!

Test your antibody or perform pre-screening before committing to a larger quantity. Sold in 10µl. Discover our selection of trial-size antibodies. Specificity confirmed

The specificity of Anti-MMP9 antibody [RM1020] (ab283575) has been confirmed by Western blot testing in MMP9 Knockout A549 cell line.

Other related products

We have a range of other formats of antibody clone [RM1020] also available for your convenience:
ab283575, Carrier free - ab283594

What are recombinant multiclonals?
Recombinant multiclonals are a mixture of recombinant antibodies co-expressed from a library of heavy and light chains. They offer several advantages including:

- The sensitivity of polyclonal antibodies by recognising multiple epitopes
- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

View our range of recombinant multiclonal antibodies.

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

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

The MMP-9 protein also known as matrix metalloproteinase-9 or gelatinase B functions as an enzyme involved in the breakdown of extracellular matrix proteins. It consists of a zinc ion at its active site and facilitates the degradation of type IV and V collagens fibronectin and elastin. MMP-9 has a molecular weight of approximately 92 kDa and is expressed in a variety of tissues including the liver lungs and immune cells. This enzyme can exist in monomer and dimer forms with its activity regulated by tissue inhibitors of metalloproteinases (TIMPs).

Biological function summary

Matrix metalloproteinase-9 profoundly influences tissue remodeling inflammation and angiogenesis. It does not form part of a larger protein complex but interacts closely with substrates in the extracellular matrix. MMP-9 plays important roles in processes such as embryogenesis reproduction and wound healing through the remodeling of surrounding tissues. The MMP-9 protein is often measured using assays like ELISA or analyzed via techniques such as Western blotting to determine its expression levels and activity in different biological contexts.

Pathways

MMP-9 participates in the matrix metalloproteinase pathway and the NF-kB signaling pathway. These pathways are essential in orchestrating processes such as tissue remodeling and inflammatory responses. MMP-9 acts alongside other matrix metalloproteinases including MMP-2 and is regulated by factors like cytokines growth factors and hormones ensuring the precision of extracellular matrix degradation during physiological processes.

Associated diseases and disorders

Matrix metalloproteinase-9 has associations with cancer invasion and metastasis as well as chronic inflammatory diseases like rheumatoid arthritis. In cancer upregulated MMP-9 expression aids tumor cells in breaching the basement membrane facilitating metastasis. The disorder-associated pathways often involve interactions with proteins like vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta). Understanding MMP-9's role in these conditions can contribute to developing targeted therapies that inhibit its activity potentially mitigating disease progression.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

16 product images

Western blot - Anti-MMP9 antibody [RM1020] (ab283575)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
MMP9 is a glycoprotein (PMID: 29329315). The 92 kDa band likely represents the zymogen prior to activating cleavage of the pro-peptide (PMID: 7688350; PMID: 12901881).
Exposure time: Lane 3-4: 3min ; Others: 37 seconds
All lanes: Western blot - Anti-MMP9 antibody [RM1020] (ab283575) at 1/1000 dilution
Lane 1: U-2 OS (Human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg
Lane 2: U-2 OS treated with 200nM TPA for 24h whole cell lysate at 20 µg
Lane 3: RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg
Lane 4: RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1ug/ml BFA for another 3h whole cell lysate at 20 µg
Lane 5: NR8383 (Rattus norvegicus lung macrophage (alveolar)) whole cell lysate at 20 µg
Lane 6: NR8383 treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1ug/ml BFA for another 3h whole cell lysate at 20 µg
Lane 7: Human lung lysate at 20 µg
Lane 8: Mouse lung lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 78 kDa
Observed band size: 84-92 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] (ab283575)
Immunohistochemical analysis of paraffin-embedded Human spleen tissue labelling MMP9 with ab283575 at 1/5000 (0.123 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the human spleen. The section was incubated with ab283575 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] (ab283575)
Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling MMP9 with ab283575 at 1/5000 (0.123 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the human tonsil. The section was incubated with ab283575 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Immunocytochemistry/ Immunofluorescence - Anti-MMP9 antibody [RM1020] (ab283575)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-2 OS cells labelling MMP9 with ab283575 at 1/50 (12.26 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).
Confocal image showing increased cytoplasmic staining in U-2 OS cells treated with 12-O-Tetradecanoylphorbol-13-acetate (200nM) for 24hours.
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.
Flow Cytometry (Intracellular) - Anti-MMP9 antibody [RM1020] (ab283575)
Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized U-2 OS (Human bone osteosarcoma epithelial cell) treated with TPA (200nM 24h) (Red) / Untreated control (Green) cells labelling MMP9 with ab283575 at 1/500 dilution (0.1ug) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat F(ab')2 Anti-Rabbit IgG(DyLight® 488, Goat F(ab')2 Anti-Rabbit IgG - H&L (DyLight® 488), pre-adsorbed ab98507) at 1/500 dilution was used as the secondary antibody.
Immunoprecipitation - Anti-MMP9 antibody [RM1020] (ab283575)
MMP9 was immunoprecipitated from 0.35 mg Mouse lung lysate 10 ug with ab283575 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283575 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: Mouse lung lysate 10 ug
Lane 2: ab283575 IP in Mouse lung lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab283575 in Mouse lung lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 min
All lanes: Immunoprecipitation - Anti-MMP9 antibody [RM1020] (ab283575)
Predicted band size: 78 kDa
Observed band size: 84-92 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] (ab283575)
Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labelling MMP9 with ab283575 at 1/5000 (0.123 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the mouse spleen. The section was incubated with ab283575 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Immunocytochemistry/ Immunofluorescence - Anti-MMP9 antibody [RM1020] (ab283575)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 cells labelling MMP9 with ab283575 at 1/50 (12.26 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).
Confocal image showing increased cytoplasmic staining in RAW 264.7 cells treated with lipopolysaccharide (100 ng/ml) for 4 hours then together with Brefeldin A (1 ug/ml) for 3 hours.
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2ug/ml) dilution.
Immunohistochemistry (Frozen sections) - Anti-MMP9 antibody [RM1020] (ab283575)
Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse lung (fresh) tissue labeling MMP9 with ab283575 at 1/100 (6.13 ug/ml) dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution (Green). The nuclear counterstain was DAPI (Blue).
Positive staining on macrophages of mouse lung is observed.
Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution.
Flow Cytometry (Intracellular) - Anti-MMP9 antibody [RM1020] (ab283575)
Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 4h then together with 1ug/ml BFA for another 3h (Right) / Untreated control (Left) cells labelling MMP9 with ab283575 at 1/500 dilution (0.1ug). A Goat F(ab')2 Anti-Rabbit IgG(DyLight® 488, Goat F(ab')2 Anti-Rabbit IgG - H&L (DyLight® 488), pre-adsorbed ab98507) at 1/500 dilution was used as the secondary antibody.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] (ab283575)
Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labelling MMP9 with ab283575 at 1/5000 (0.123 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the rat spleen. The section was incubated with ab283575 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Immunohistochemistry (Frozen sections) - Anti-MMP9 antibody [RM1020] (ab283575)
Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat lung (fresh) tissue labeling MMP9 with ab283575 at 1/100 (6.13 ug/ml) dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution (Green). The nuclear counterstain was DAPI (Blue).
Positive staining on macrophages of rat lung is observed.
Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution.
Indirect ELISA - Anti-MMP9 antibody [RM1020] (ab283575)
ELISA using ab283575 at varying antibody concentrations and antigen concentration at 1000 ng/ml. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] (ab283575)
Immunohistochemical analysis of paraffin-embedded human gastric carcinoma tissue labelling MMP9 with ab283575 at 1:5000 dilution (0.123 μg/ml), followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on immune cells of human gastric carcinoma is observed. The section was incubated with ab283575 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.
Western blot - Anti-MMP9 antibody [RM1020] (ab283575)
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).
Blocking and diluting buffer and concentration: 5% NFDM/TBST
All lanes: Western blot - Anti-MMP9 antibody [RM1020] (ab283575) at 1/1000 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 100ng/ml TPA for 24 hours, then together with 1ug/ml BFA for another 3h whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Exposure time: 60s
Western blot - Anti-MMP9 antibody [RM1020] (ab283575)
MMP9 Western blot staining using rabbit Anti-MMP9 antibody
Western blot: Rabbit Recombinant Multiclonal[RM1020] to MMP9 283575 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (Anti-GAPDH antibody [6C5] - Loading Control 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 [RM1020] (ab283575) 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
Performed under reducing conditions.
Predicted band size: 78 kDa
Observed band size: 90 kDa