Flow cytometry overlay histogram showing wild-type A431 (green line) and EPCAM knockout A431 cells stained with ab223582 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab223582) (1x10⁶ in 100μl at 0.2 μg/ml) for 30 min at 4°C.

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 4°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type A431 - black line; EPCAM knockout A431 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

All lanes : Anti-EpCAM antibody [EPR20532-225] (ab223582) at 1/1000 dilution

Lane 1 : Wild-type A431 whole cell lysate
Lane 2 : EPCAM knockout A431 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 35 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Lanes 1 - 3: Merged signal (red and green). Green - ab223582 observed at 40 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab223582 was shown to react with EpCAM in A431 wild-type cells in Western blot. Loss of signal was observed when EpCAM knockout sample was used. A431 wild-type and EpCAM knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab223582 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling EpCAM with ab223582 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on human colon is observed (PMID: 15637741). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ab223582 staining EpCAM in wild-type A431 cells (top panel) and EpCAM knockout A431 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% PBS-Tween 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 ab223582 at 0.1μg/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. This antibody performed similarly using 100% methanol fixation. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

Flow cytometric analysis of HCT 116 (human colorectal carcinoma cell line) cell line labeling EpCAM with ab223582 at 1/500 (red) compared with a rabbit monoclonal IgG Isotype control (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.

Total viable cells were gated for the final data.

ab223582 staining EpCAM in T47D positive cells (top panel) and HeLa negative cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% PBS-Tween 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 ab223582 at 0.1μg/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. This antibody performed similarly using 100% methanol fixation. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

ab223582 staining EpCAM in wild-type A431 cells (top panel) and EpCAM knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% PBS-Tween 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 ab223582 at 1/5000 dilution 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).

Immunofluorescent analysis of 4% paraformaledehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (human colorectal carcinoma cell line) cells labeling EpCAM with ab223582 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 HT-29 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.

All lanes : Anti-EpCAM antibody [EPR20532-225] (ab223582) at 1/5000 dilution

Lane 1 : HCT 116 (human colorectal carcinoma cell line) lysate
Lane 2 : HT-29 (human colorectal adenocarcinoma cell line) lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 35 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-EpCAM antibody [EPR20532-225] (ab223582) at 1/1000 dilution

Lane 1 : Human breat cancer lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lane 2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Predicted band size: 35 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human thyroid cancer tissue labeling EpCAM with ab223582 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on human thyroid cancer is observed (PMID: 15637741). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaledehyde-fixed, 0.1% Triton X-100 permeabilized HT-29 (human colorectal adenocarcinoma cell line) cells labeling EpCAM with ab223582 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 HT-29 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.

EpCAM was immunoprecipitated from 0.35 mg of HCT 116 (human colorectal carcinoma cell line) whole cell lysate with ab223582 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab223582 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HCT 116 lysate 10 μg (Input).

Lane 2: ab223582 IP in HCT 116 lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab223582 in HCT 116 whole cell lysate.

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

Exposure time: 30 seconds.