Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling CEACAM6 with ab275022 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human colon cancer (PMID: 27284373). The section was incubated with ab275022 for 30 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 ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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% Triton X-100 permeabilized BxPC-3 BxPC-3 (human pancreas adenocarcinoma cell) cells labelling CEACAM6 with ab275022 at 1/500 (1.048 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous and cytoplamic staining in BxPC-3 cell line. Low expression control: LNCaP: PMID: 16204051. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

CEACAM6 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate with ab275022 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275022 at 1/2000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

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

Lane 2: ab275022 IP in A549 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab275022 in A549 whole cell lysate

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

Exposure time: 3 seconds.

 

Flow cytometric analysis of LNCaP (human prostate carcinoma epithelial cell, Left) / BxPC-3 (human pancreas adenocarcinoma epithelial cell, Right) cells labelling CEACAM6 with ab275022 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: LNCaP.

Gated on viable cells.

All lanes : Anti-CEACAM6 antibody [EPR23956-80] (ab275022) at 1/1000 dilution

Lane 1 : His-tagged human CEACAM6 recombinant protein, 20ng
Lane 2 : His-tagged human CEACAM1 recombinant protein (aa35-428), 20ng
Lane 3 : His-tagged human CEACAM5 recombinant protein (aa35-685), 20ng

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 37 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 15 seconds.

The other family members have lower homology than the tested proteins.

 

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling CEACAM6 with abab275022 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human spleen. The section was incubated with ab275022 for 30 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 ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Anti-CEACAM6 antibody [EPR23956-80] (ab275022) at 1/1000 dilution + Human lung tissue lysate at 20 µg

Secondary
VeriBlot for IP secondary antibody(HRP)(ab131366) at 1/1000 dilution

Predicted band size: 37 kDa
Observed band size: 40-100 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 26 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 19244123, 28892050, 16204051).

All lanes : Anti-CEACAM6 antibody [EPR23956-80] (ab275022) at 1/1000 dilution

Lane 1 : A549 (human lung carcinoma epithelial cell) whole cell lysate
Lane 2 : LNCaP (human prostate carcinoma epithelial cell) whole cell lysate
Lane 3 : BxPC-3 (human pancreas epithelial adenocarcinoma cell) whole cell lysate
Lane 4 : LoVo (human colorectal adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 37 kDa
Observed band size: 40-100 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-2: 15 seconds; Lane3: 3 seconds; Lane 4: 26 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 19244123, 28892050, 16204051).

Low expression control: LNCaP (PMID: 16204051).

CEACAM6 was immunoprecipitated from 0.35 mg BxPC-3 (human pancreas adenocarcinoma epithelial cell) whole cell lysate with ab275022 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275022 at 1/2000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: BxPC-3 (human pancreas adenocarcinoma epithelial cell) whole cell lysate 10 ug

Lane 2: ab275022 IP in BxPC-3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab275022 in BxPC-3 whole cell lysate

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

Exposure time: 3 seconds.