Anti-MCP1 antibody [EPR21025]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [EPR21025] (AB214819)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [EPR21025] (AB214819)

Immunohistochemical analysis of paraffin-embedded wild-type HeLa cell pellets 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [EPR21025] (AB214819)

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.

• ICC

Lab

Immunocytochemistry - Anti-MCP1 antibody [EPR21025] (AB214819)

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).

• Flow Cyt

Lab

Flow Cytometry - Anti-MCP1 antibody [EPR21025] (AB214819)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MCP1 antibody [EPR21025] (AB214819)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [EPR21025] (AB214819)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-MCP1 antibody [EPR21025] (AB214819)

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.

All lanes:

Immunoprecipitation - Anti-MCP1 antibody [EPR21025] (ab214819)

Predicted band size: 11 kDa,14 kDa

Observed band size: 12 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-MCP1 antibody [EPR21025] (AB214819)

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

All lanes:

Western blot - Anti-MCP1 antibody [EPR21025] (ab214819)

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Wild-type A549 Treated BFA (1 ug/mL, 3 h) cell lysate, at 20 µg

Lane 3:

CCL2 knockout A549 Treated BFA (1 ug/mL, 3 h) cell lysate at 20 µg

Observed band size: 13 kDa

false

• WB

Lab

Western blot - Anti-MCP1 antibody [EPR21025] (AB214819)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MCP1 antibody [EPR21025] (ab214819) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia cell line) whole cell lysate at 20 µg

Lane 2:

THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, <a href='/en-us/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>) 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 at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 11 kDa

Observed band size: 11 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-MCP1 antibody [EPR21025] (AB214819)

False 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 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.

All lanes:

Western blot - 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 at 20 µg

Lane 2:

Wild-type HeLa Treated TNF alpha (20ng/mL, 6h) + Brefeldin A (1μg/ml,3h) cell lysate at 20 µg

Lane 3:

CCL2 knockout HeLa Vehicle Control TNF alpha (0ng/mL, 6h) + Brefeldin A (1μg/ml,3h) cell lysate at 20 µg

Lane 4:

CCL2 knockout HeLa Treated TNF alpha (20ng/mL, 6h) + Brefeldin A (1μg/ml,3h) cell lysate at 20 µg

Predicted band size: 11 kDa

Observed band size: 11 kDa

false