Anti-Carcino Embryonic Antigen CEA抗体(ab131070)

Immunohistochemical analysis of paraffin-embedded Human colon cancer tissue labeling Carcino Embryonic Antigen CEA with ab131070 at 1 ug/ml. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C.
The section was blocked with 10% goat serum then incubated with ab131070 overnight at 4°C.
The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.
Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution).

Western blot: Anti-CEACAM5 antibody (ab131070) staining at 0.1 ug/ml, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab131070 was shown to bind specifically to CEACAM5. A band was observed at 170 kDa in wild-type A549 cell lysates with no signal observed at this size in CEACAM5 knockout cell line. To generate this image, wild-type and CEACAM5 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 3 % 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 and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. Proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at room temperature. The membrane was incubated with primary antibody overnight at 4°C, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with secondary antibody for 1.5 hour at room temperature.