Recombinant Anti-MCP1 antibody [EPR21025] (ab214819)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR21025] to MCP1
- Suitable for: ICC/IF, Flow Cyt (Intra), IHC-P, WB, IP
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-MCP1 antibody [EPR21025]
See all MCP1 primary antibodies -
Description
Rabbit monoclonal [EPR21025] to MCP1 -
Host species
Rabbit -
Specificity
Stimulation may be required for the detection of MCP1, as it is not constitutively expressed.
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Tested applications
Suitable for: ICC/IF, Flow Cyt (Intra), IHC-P, WB, IPmore details -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: THP-1 treated with PMA (ab120297), LPS, and BFA whole cell lysate. ICC/IF: THP-1 cells treated with PMA (ab120297), LPS, and BFA, wild-type and MCP1 knockout HeLa cells (ab255372) treated with TNF-alpha (20ng/mL, 6 hours). Flow Cyt (intra): THP-1 treated with PMA (ab120297), LPS, and BFA. IP: THP-1 treated with PMA (ab120297), LPS, and BFA whole cell lysate. IHC: Human lung adenocarcinoma; THP-1 cell pellets treated with PMA, LPS, and BFA; Wild type HeLa cell pellets treated with BFA, and TNF-alpha.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR21025 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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KO cell lines
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab214819 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 |
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ICC/IF |
1/50.
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Flow Cyt (Intra) |
1/500.
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IHC-P |
1/2000 - 1/10000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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WB |
1/1000. Detects a band of approximately 11 kDa (predicted molecular weight: 11 kDa).
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IP |
1/30.
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Notes |
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ICC/IF
1/50. |
Flow Cyt (Intra)
1/500. |
IHC-P
1/2000 - 1/10000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
WB
1/1000. Detects a band of approximately 11 kDa (predicted molecular weight: 11 kDa). |
IP
1/30. |
Target
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Function
Chemotactic factor that attracts monocytes and basophils but not neutrophils or eosinophils. Augments monocyte anti-tumor activity. Has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, like psoriasis, rheumatoid arthritis or atherosclerosis. May be involved in the recruitment of monocytes into the arterial wall during the disease process of atherosclerosis. -
Sequence similarities
Belongs to the intercrine beta (chemokine CC) family. -
Post-translational
modificationsProcessing at the N-terminus can regulate receptor and target cell selectivity. Deletion of the N-terminal residue converts it from an activator of basophil to an eosinophil chemoattractant. -
Cellular localization
Secreted. - Information by UniProt
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Database links
- Entrez Gene: 6347 Human
- Omim: 158105 Human
- SwissProt: P13500 Human
- Unigene: 303649 Human
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Alternative names
- C-C motif chemokine 2 antibody
- CCL2 antibody
- CCL2_HUMAN antibody
see all
Images
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All lanes : Anti-MCP1 antibody [EPR21025] (ab214819) at 1/1000 dilution
Lane 1 : Untreated THP-1 (human monocytic leukemia cell line) whole cell lysate
Lane 2 : THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours, then treated with 100ng/ml lipopolysaccharide (LPS) for 7 hours, then with 1 µg/ml Brefeldin A (BFA) added after 4 hours, whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 11 kDa
Observed band size: 11 kDa
Exposure time: 3 minutesBlocking/Dilution buffer: 5% NFDM/TBST.
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Immunohistochemical analysis of paraffin-embedded wild-type and CCL2 knockout HeLa cell pellets; labelling MCP1 with ab214819 at a 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) secondary antibody. Counter stained with Hematoxylin.
Image A: Wild-type Hela cell pellets treated with Brefeldin A (1μg/ml) for 3 hours.
Image B: Wild-type Hela cell pellets treated with TNF-alpha (TNFα, 20ng/ml) for 3 hours, 1µg/ml Brefeldin A was added for additional 3 hours.
Image C: CCL2 knockout HeLa cell pellets treated with Brefeldin A (1μg/ml) for 3 hours.
Image D: CCL2 knockout HeLa cell pellets treated with TNF-alpha (TNFα, 20ng/ml) for 3 hours, 1µg/ml Brefeldin A was added for additional 3 hours.Positive staining on wild-type HeLa cell pellets treated with Brefeldin A (1μg/ml) for 3 hours (Image A) and wild-type HeLa cell pellets treated with TNF-alpha (TNFα, 20ng/ml) for 3 hours, 1µg/ml Brefeldin A was added for additional 3 hours (Image B); No staining on CCL2 knockout HeLa cell pellets treated with Brefeldin A (1μg/ml) for 3 hours (Image C) and CCL2 knockout HeLa cell pellets treated with TNF-alpha (TNFα, 20ng/ml) for 3 hours, 1µg/ml Brefeldin A was added for additional 3 hours (Image D). The section was incubated with ab214819 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.
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ab214819 staining MCP1 in wild-type and MCP1 knockout HeLa cells (ab255372), untreated or treated with TNF-alpha (20ng/ml, 6 hours) and Brefeldin A (1μg/mL, 3 hours). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab214819 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 0.1% Tween-20-permeabilized THP-1 (human monocytic leukemia cell line) cell line, treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1µg/ml Brefeldin A (BFA) for another 3h (Right) / Untreated control (Left)labeling MCP1 with ab214891 at 1/500 dilution. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.
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MCP1 was immunoprecipitated from 0.35 mg of THP-1 (human monocytic leukemia cell line) treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate with ab214819 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab214819 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1,000 dilution
Lane 1: THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate 10 µg (Input).
Lane 2: ab214819 IP in THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab214819 in THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate.
Blocking and dilution buffer: 5% NFDM/TBST.
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All lanes : Anti-MCP1 antibody [EPR21025] (ab214819) at 1/400 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : Wild-type A549 Treated BFA (1 ug/mL, 3 h) cell lysate
Lane 3 : CCL2 knockout A549 Treated BFA (1 ug/mL, 3 h) cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 11 kDa
Observed band size: 13 kDa why is the actual band size different from the predicted?Anti-CCL2 antibody [EPR21025] (ab214819) staining at 1/400 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab214819 was shown to bind specifically to CCL2. A band was observed at 13 kDa in wild-type A549 cell lysates with no signal observed at this size in CCL2 knockout cell line ab270478 (knockout cell lysate ab270501). To generate this image, wild-type and CCL2 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 5 % 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 at 1/20000 dilution and Goat anti-Mouse IgG H&L 680RD at 1/80000 dilution.
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Immunohistochemical analysis of paraffin-embedded THP-1 (human monocytic leukemia monocyte) cell pellets labelling MCP1 with ab214819 at a 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) secondary antibody. Counter stained with Hematoxylin.
Image A: THP-1 (human monocytic leukemia monocyte) cell pellets treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was added for additional 3 hours.
Image B: Untreated THP-1 cell pellets.Cytoplasmic staining on THP-1 cell pellets treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was added for additional 3 hours (Image A); No staining on untreated THP-1 cell pellets (Image B). The section was incubated with ab214819 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.
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Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (human monocytic leukemia cell line) cells, untreated or treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours, then treated with 100ng/ml lipopolysaccharide (LPS) for 7 hours, with 1 μg/ml Brefeldin A (BFA) added after 4 hours, labeling MCP1 with ab214819 at 1/50 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in THP-1 treated cells.
The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.
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All lanes : Anti-MCP1 antibody [EPR21025] (ab214819) at 1/1000 dilution
Lane 1 : Wild-type HeLa Vehicle Control TNF alpha (0ng/mL, 6h) + Brefeldin A (1µg/ml,3h) cell lysate
Lane 2 : Wild-type HeLa Treated TNF alpha (20ng/mL, 6h) + Brefeldin A (1µg/ml,3h) cell lysate
Lane 3 : CCL2 knockout HeLa Vehicle Control TNF alpha (0ng/mL, 6h) + Brefeldin A (1µg/ml,3h) cell lysate
Lane 4 : CCL2 knockout HeLa Treated TNF alpha (20ng/mL, 6h) + Brefeldin A (1µg/ml,3h) cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 11 kDaFalse colour image of Western blot: Anti-MCP1 antibody [EPR21025] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab214819 was shown to bind specifically to MCP1. A band was observed at 11 kDa in wild-type HeLa cell lysates with no signal observed at this size in ccl2 knockout cell line ab255372 (knockout cell lysate ab263807). To generate this image, wild-type and ccl2 knockout y cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma sections labelling MCP1 with ab214819 at a 1/2000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) secondary antibody. Counter stained with Hematoxylin.
Positive staining on human lung adenocarcinoma. The section was incubated with ab214819 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.
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Immunohistochemical analysis of paraffin-embedded human skeletal muscle sections labelling MCP1 with ab214819 at a 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) secondary antibody. Counter stained with Hematoxylin.
Negative control: no staining on human skeletal muscle.
The section was incubated with ab214819 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (6)
ab214819 has been referenced in 6 publications.
- Huang WC et al. Endurance exercise ameliorates Western diet-induced atherosclerosis through modulation of microbiota and its metabolites. Sci Rep 12:3612 (2022). PubMed: 35256637
- Ding J et al. Role of PIM2 in acute lung injury induced by sepsis. Exp Ther Med 24:543 (2022). PubMed: 35978927
- Jiang H & Lv J MicroRNA-301a-3p increases oxidative stress, inflammation and apoptosis in ox-LDL-induced HUVECs by targeting KLF7. Exp Ther Med 21:569 (2021). PubMed: 33850541
- Zhao H & He Y The Inhibitory Effect of Lysophosphatidylcholine on Proangiogenesis of Human CD34+ Cells Derived Endothelial Progenitor Cells. Front Mol Biosci 8:682367 (2021). PubMed: 34179086
- Zheng Z et al. Hippo-YAP/MCP-1 mediated tubular maladaptive repair promote inflammation in renal failed recovery after ischemic AKI. Cell Death Dis 12:754 (2021). PubMed: 34330891
- Dikalova AE et al. Mitochondrial Deacetylase Sirt3 Reduces Vascular Dysfunction and Hypertension While Sirt3 Depletion in Essential Hypertension Is Linked to Vascular Inflammation and Oxidative Stress. Circ Res 126:439-452 (2020). PubMed: 31852393