Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)

Rabbit Recombinant Multiclonal COX2 / Cyclooxygenase 2 antibody. Suitable for IP, WB, IHC-P, ICC/IF and reacts with Mouse, Human, Rat samples. Cited in 3 publications.

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Images

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574), 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.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

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

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/30	Notes -
Species Human	Dilution info 1/30	Notes -

Expected
Expected

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

Tested
Tested

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

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse, Rat	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 Rat, Mouse, Human	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Rat, Mouse, Human	Dilution info -	Notes -

Target data

See full target information Ptgs2

Function

Dual cyclooxygenase and peroxidase in the biosynthesis pathway of prostanoids, a class of C20 oxylipins mainly derived from arachidonate, with a particular role in the inflammatory response (PubMed:12925531, PubMed:20463020, PubMed:20810665, PubMed:21489986, PubMed:22942274). The cyclooxygenase activity oxygenates arachidonate (AA, C20:4(n-6)) to the hydroperoxy endoperoxide prostaglandin G2 (PGG2), and the peroxidase activity reduces PGG2 to the hydroxy endoperoxide PGH2, the precursor of all 2-series prostaglandins and thromboxanes. This complex transformation is initiated by abstraction of hydrogen at carbon 13 (with S-stereochemistry), followed by insertion of molecular O2 to form the endoperoxide bridge between carbon 9 and 11 that defines prostaglandins. The insertion of a second molecule of O2 (bis-oxygenase activity) yields a hydroperoxy group in PGG2 that is then reduced to PGH2 by two electrons (PubMed:12925531, PubMed:20463020, PubMed:20810665, PubMed:21489986, PubMed:22942274). Similarly catalyzes successive cyclooxygenation and peroxidation of dihomo-gamma-linoleate (DGLA, C20:3(n-6)) and eicosapentaenoate (EPA, C20:5(n-3)) to corresponding PGH1 and PGH3, the precursors of 1- and 3-series prostaglandins (By similarity). In an alternative pathway of prostanoid biosynthesis, converts 2-arachidonoyl lysophopholipids to prostanoid lysophopholipids, which are then hydrolyzed by intracellular phospholipases to release free prostanoids (By similarity). Metabolizes 2-arachidonoyl glycerol yielding the glyceryl ester of PGH2, a process that can contribute to pain response (By similarity). Generates lipid mediators from n-3 and n-6 polyunsaturated fatty acids (PUFAs) via a lipoxygenase-type mechanism. Oxygenates PUFAs to hydroperoxy compounds and then reduces them to corresponding alcohols (By similarity). Plays a role in the generation of resolution phase interaction products (resolvins) during both sterile and infectious inflammation. Metabolizes docosahexaenoate (DHA, C22:6(n-3)) to 17R-HDHA, a precursor of the D-series resolvins (RvDs). As a component of the biosynthetic pathway of E-series resolvins (RvEs), converts eicosapentaenoate (EPA, C20:5(n-3)) primarily to 18S-HEPE that is further metabolized by ALOX5 and LTA4H to generate 18S-RvE1 and 18S-RvE2. In vascular endothelial cells, converts docosapentaenoate (DPA, C22:5(n-3)) to 13R-HDPA, a precursor for 13-series resolvins (RvTs) shown to activate macrophage phagocytosis during bacterial infection (By similarity). In activated leukocytes, contributes to oxygenation of hydroxyeicosatetraenoates (HETE) to diHETES (5,15-diHETE and 5,11-diHETE) (By similarity). Can also use linoleate (LA, (9Z,12Z)-octadecadienoate, C18:2(n-6)) as substrate and produce hydroxyoctadecadienoates (HODEs) in a regio- and stereospecific manner, being (9R)-HODE ((9R)-hydroxy-(10E,12Z)-octadecadienoate) and (13S)-HODE ((13S)-hydroxy-(9Z,11E)-octadecadienoate) its major products (By similarity). During neuroinflammation, plays a role in neuronal secretion of specialized preresolving mediators (SPMs) 15R-lipoxin A4 that regulates phagocytic microglia (PubMed:29662056).

Additional Targets

Ptgs2, Ptgs2

Alternative names

Cox-2, Cox2, Pghs-b, Tis10, Ptgs2, Prostaglandin G/H synthase 2, Cyclooxygenase-2, Glucocorticoid-regulated inflammatory cyclooxygenase, Gripghs, Macrophage activation-associated marker protein P71/73, PES-2, PHS II, Prostaglandin H2 synthase 2, Prostaglandin-endoperoxide synthase 2, TIS10 protein, COX-2, PGH synthase 2, PGHS-2

Recommended products

Rabbit Recombinant Multiclonal COX2 / Cyclooxygenase 2 antibody. Suitable for IP, WB, IHC-P, ICC/IF and reacts with Mouse, Human, Rat samples. Cited in 3 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
Immunogens: The exact immunogen used to generate this antibody is proprietary information.
Clone number: RM1026
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

This product is a recombinant multiclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

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

Cyclooxygenase 2 also known as COX2 is an enzyme involved in the conversion of arachidonic acid to prostaglandins which are lipid compounds with hormone-like effects. It has alternative names including prostaglandin-endoperoxide synthase 2. The molecular weight of COX2 is approximately 72 kDa. This enzyme is expressed in various tissues including the brain kidneys and areas of inflammation. COX2 expression increases during inflammatory responses and is induced by pro-inflammatory cytokines.

Biological function summary

COX2 plays a significant role in the inflammatory response and is part of the complex process of synthesizing prostaglandins. These compounds mediate inflammation and pain making COX2 an important target for understanding these processes. COX2 is not ubiquitously expressed but rather is induced in activated macrophages and other cells during inflammatory conditions. Its function is also important for normal physiological processes like ovulation and implantation.

Pathways

COX2 is essential in the prostaglandin biosynthesis pathway connecting it to the arachidonic acid metabolism pathway. Cyclooxygenase 2 works with phospholipase A2 which releases arachidonic acid from the phospholipid membrane. COX2 then converts this acid to prostaglandin H2 a precursor for other prostaglandins. COX1 the other isoform of cyclooxygenase is closely related to COX2 and while they have different expression patterns they share some functional similarities in these pathways.

Associated diseases and disorders

COX2 is connected to inflammatory conditions like arthritis and cancer. Its expression often increases in various cancer types contributing to tumor growth and metastasis by promoting angiogenesis and suppressing immune responses. The enzyme is also linked to rheumatoid arthritis where its overexpression exacerbates inflammation. COX2 inhibitors like ketorolac tromethamine or naproxen structure mitigate symptoms by decreasing prostaglandin synthesis. These inhibitors also interact with COX1 but selective inhibition of COX2 targets inflammation more effectively with fewer gastric side effects associated with COX1 inhibition.

Product promise

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11 product images

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Negative control: MCF7 (PMID: 24325753, PMID: 16997132)
Lysates/proteins at 20 μg per lane.
Performed under reducing conditions.
False colour image of Western blot: Anti- COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab283574 was shown to bind specifically to COX2 / Cyclooxygenase 2. Target band was observed at 74 kDa in wild-type A549 cell lysates with no signal observed at this size in COX2 / Cyclooxygenase 2 knockout cell line Human PTGS2 (COX2 / Cyclooxygenase 2) knockout A549 cell line ab280802. To generate this image, wild-type and COX2 / Cyclooxygenase 2 knockout A549 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) at 1/10000 dilution
Lane 1: PTGS2 (COX2 / Cyclooxygenase 2) KO A549 (Human lung carcinoma epithelial cell) cell lysate at 20 µg with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 2: Wild-type A549 cell lysate at 20 µg with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 3: U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 4: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Secondary
All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 69 kDa
Observed band size: 74 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Immunohistochemical analysis of paraffin-embedded human colon tissue labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/500 dilution (1.058 μg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on human colon. The section was incubated with ab283574 overnight at 4°C. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).
Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0)
Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
Negative control: MCF7 (PMID: 24325753, PMID: 16997132)
Lower bands could be COX-2 fragments due to proteolysis. (PMID: 32366045)
Exposure time: Lane 1-4: 2 min Lane 5-6: 3 min
All lanes: Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) at 1/1000 dilution
Lane 1: U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg
Lane 2: MCF7 (Human breast adenocarcinoma epithelial cell) 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 1 µg/ml lipopolysaccharide (LPS) for 6h whole cell lysate at 20 µg
Lane 5: C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg
Lane 6: C6 treated with 100 ng/ml lipopolysaccharide (LPS) for 4h 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 1/100000 dilution
Predicted band size: 69 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Immunohistochemical analysis of paraffin-embedded human liver tissue labeling COX2 / Cyclooxygenase 2 with ab283574 at 1/500 dilution (1.058 μg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on human colon liver. The section was incubated with ab283574 overnight at 4°C. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).
Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/500 dilution (1.058 μg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on human colon carcinoma. The section was incubated with ab283574 overnight at 4°C. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).
Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0)
Immunocytochemistry/ Immunofluorescence - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/1000 dilution (0.529 μg/ml), 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 dilution (2 μg/ml)(Green). Confocal image showing cytoplasmic staining in RAW 264.7 cell line after treatment with lipopolysaccharide (1 μg/ml) for 6 hours is observed. 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 dilution (2.5 μg/ml)(Red). The Nuclear counterstain was DAPI (Blue).
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 dilution (2 μg/ml).
Immunoprecipitation - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
COX2 / Cyclooxygenase 2 was immunoprecipitated from 0.35 mg U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate with ab283574 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283574 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: U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate 10μg
Lane 2: ab283574 IP in U-87 MG whole cell lysate
Lane 3:Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab283574 in U-87 MG whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 6 seconds
Lower bands could be COX-2 fragments due to proteolysis. (PMID: 32366045)
All lanes: Immunoprecipitation - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Predicted band size: 69 kDa
Immunocytochemistry/ Immunofluorescence - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-87 MG (human glioblastoma-astrocytoma epithelial cell) cells labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/500 dilution (1.058 μg/ml), 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 dilution (2 μg/ml)(Green). Confocal image showing cytoplasmic staining in of U-87 MG cell line. Negative control: MCF7 (PMID:18199541) is observed. 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 dilution (2.5 μg/ml)(Red). The Nuclear counterstain was DAPI (Blue).
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 dilution (2 μg/ml).
Immunoprecipitation - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
COX2 / Cyclooxygenase 2 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1 μg/ml LPS for 6h whole cell lysate with ab283574 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283574 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: RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml LPS for 6h whole cell lysate 10μg
Lane 2: ab283574 IP in RAW 264.7 treated with 1 μg/ml LPS for 6h whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab283574 in RAW 264.7 treated with 1 μg/ml LPS for 6h whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 6 seconds
All lanes: Immunoprecipitation - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Predicted band size: 69 kDa
Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
Compared with Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] ab179800, ab283574 has higher sensitivity, we recommend ab283574 as an alternative for testing COX2 in western blot.
Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a loading control at a 1/1000000 dilution.
Blocking and dilution buffer: 5% NFDM/TBST.
Lanes 1 - 3: Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] ab179800) at 1/1000 dilution
Lanes 4 - 6: Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) at 1/1000 dilution
Lanes 1 and 3: Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lanes 2 and 4: PTGS2 knockout HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lanes 3 and 6: A549 (Human lung carcinoma epithelial cell) 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
Predicted band size: 69 kDa
Observed band size: 75 kDa
Exposure time: 180s
Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)
False colour image of Western blot: Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab283574 was shown to bind specifically to COX2 / Cyclooxygenase 2. A band was observed at 74-76 kDa in wild-type A549 cell lysates with no signal observed at this size in PTGS2 knockout cell line Human PTGS2 (COX2 / Cyclooxygenase 2) knockout A549 cell line ab280802 (knockout cell lysate ab283825). Band at 70 kDa in both wild-type and knockout samples is non-specific but exact protein is not determined. To generate this image, wild-type and PTGS2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: PTGS2 knockout A549 cell lysate at 20 µg
Lane 3: U-87 MG cell lysate at 20 µg
Lane 4: MOLT-4 cell lysate at 20 µg
Performed under reducing conditions.
Observed band size: 74-76 kDa