Recombinant Anti-CEACAM1 antibody [EPR26490-88] (ab300061)

Lanes 1-3: Merged signal (red and green). Green - ab300061 observed at 100-180 kDa. Red - loading control ab8245 observed at 36 kDa.

ab300061 Anti-CEACAM1 antibody [EPR26490-88] was shown to specifically react with CEACAM1 in wild-type A549 cells (ab255450). Loss of signal was observed when knockout cell line (ab267047) was used. Wild-type and CEACAM1 knockout samples were subjected to SDS-PAGE. ab300061 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4? overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

Blocking and dilution buffer and concentration: 5% NFDM/TBST. 

Exposure time: Lanes 1-3:10 seconds; Lane 4: 48 seconds. 

Blocking and dilution buffer and concentration: 5% NFDM/TBST. 

Exposure time: Lanes 1-3:10 seconds; Lane 4: 48 seconds. 

Immunohistochemical analysis of paraffin-embedded human colon tissue labelling CEACAM1 with ab300061 at 1/2000 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human colon (PMID: 8543780). The section was incubated with ab300061 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody (ab300061) followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labelling CEACAM1 with ab300061 at 1/2000 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human endometrium (PMID: 9626043). The section was incubated with ab300061 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody (ab300061) followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded human liver tissue labelling CEACAM1 with ab300061 at 1/2000 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human liver (PMID: 27377843). The section was incubated with ab300061 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody (ab300061) followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded human hepatic carcinoma tissue labelling CEACAM1 with ab300061 at 1/2000 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human hepatic carcinoma (PMID: 27377843). The section was incubated with ab300061 for 30 mins at room temperature The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody (ab300061) followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeilized CEACAM1 KO A549 (CEACAM1 knockout human lung carcinoma epithelial cell) (ab267047) cells lebelling CEACAM1 with ab300061 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing membrane and cytoplasmic staining in parental A549 cells, no staining in CEACAM1 KO A549 cells.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

Flow cytometric analysis of Wild-type A549 (Human lung carcinoma epithelial cell, Right)/ CEACAM1 knockout A549 cells (Left) cells labelling CEACAM1 with ab300061 at 1/500 dilution/ Left and Right compared with a isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti-Rabbit IgG (Alexa Fluor^® 647, ab150083) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells. Positive staining on A549 cells (ab255450), while no staining on CEACAM1 knockout A549 cells (ab267047).

CEACAM1 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate 10 µg with ab300061 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab300061 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: A549 (human lung carcinoma epithelial cell) whole cell lysate 10 µg (Input)

Lane 2: ab300061 IP in A549 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300061 in A549 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 32 seconds