Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Carcino Embryonic Antigen CEA with ab207718 at 1/40,000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in human colon (PMID: 25042385) is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-CEACAM5 + CD66b + CEACAM1 + CEACAM6 antibody [EPR20721] (ab207718) at 1/1000 dilution

Lane 1 : His-GST-tagged human CEACAM5 recombinant protein (aa238-686) 10 ng
Lane 2 : His-tagged human CEACAM6 recombinant protein (aa35-326) 10 ng
Lane 3 : His-tagged human CEACAM8 recombinant protein (aa35-335) 10 ng
Lane 4 : His-tagged human CEACAM1 recombinant protein (aa35-428) 10 ng

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 76 kDa
Observed band size: 30,50,75 kDa why is the actual band size different from the predicted?
Additional bands at: 32 kDa (possible post-translational modification)

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

Exposure times:

Lanes 1-3: 10 seconds

Lane 4: 4 minutes

Observed MW: 50, 30, 32, 75 kDa.

The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument.

All lanes : Anti-CEACAM5 + CD66b + CEACAM1 + CEACAM6 antibody [EPR20721] (ab207718) at 1/1000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : PANC-1 (human pancreatic epithelioid carcinoma epithelial cell), 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

Developed using the ECL technique.

Predicted band size: 76 kDa
Observed band size: 110-150,75-90 kDa why is the actual band size different from the predicted?


Exposure time: 58 seconds

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

Observed MW: 110-150, 75-90  kDa.

The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument.

All lanes : Anti-CEACAM5 + CD66b + CEACAM1 + CEACAM6 antibody [EPR20721] (ab207718) at 1/1000 dilution

Lane 1 : Caco-2 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 10 µg
Lane 2 : SW480 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 10 µg
Lane 3 : Human colon lysate at 20 µg
Lane 4 : LoVo (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 76 kDa
Observed band size: 110-150,75-90 kDa why is the actual band size different from the predicted?

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

Exposure times:

Lanes 1-2: 3 minutes

Lanes 3-4: 10 seconds

Observed MW 110-150, 75-90 kDa.

The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument. The expression profile observed is consistent with differential glycosylation of this protein (PMID: 1717152).

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling Carcino Embryonic Antigen CEA with ab207718 at 1/40,000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in human colon cancer (PMID: 25042385) is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling Carcino Embryonic Antigen CEA with ab207718 at 1/40,000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining of spleen-infiltrating neutrophils (PMID: 22064717 and 19077207) is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 100% methanol fixed MCF7 (human breast adenocarcinoma epithelial cell) cells labeling Carcino Embryonic Antigen CEA with ab207718 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membranous staining on MCF7 cell line.

Negative control for cells: PANC-1 .

Counterstained with ab195889 anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red) and DAPI (blue).

The negative control is the secondary antibody only.

Flow cytometric analysis of PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) (Left) / MCF7 (human breast adenocarcinoma epithelial cell) (Right) labeling Carcino Embryonic Antigen CEA with ab207718 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled 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. Cells were not fixed or permeabilized. Gated on total viable cells.