Anti-EpCAM antibody [EPR20532-222]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Immunofluorescent analysis of 100% methanol-fixed HCT 116 (Human colorectal carcinoma cell line) cells labeling EpCAM with ab213500 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membranous staining on HCT 116 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling EpCAM with ab213500 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab214880) Ready to use. Counter stained with Hematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab214880). Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. Positive staining on human breast carcinoma. The section was incubated with ab213500 at 4°C overnight.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling EpCAM with ab213500 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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) (ab97051) at 1/500 dilution.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Immunohistochemical analysis of paraffin-embedded human endometrium cancer tissue labeling EpCAM with ab213500 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membranous staining on tumor cells of human endometrium 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) (ab97051) at 1/500 dilution.

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Immunofluorescent analysis of 100% methanol-fixed 4T1 (Mouse mammary gland carcinoma cell line) cells labeling EpCAM with ab213500 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membranous staining on 4T1 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling EpCAM with ab213500 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membranous staining on rat 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) (ab97051) at 1/500 dilution.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling EpCAM with ab213500 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membranous staining on mouse 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) (ab97051) at 1/500 dilution.

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

• WB

Supplier Data

Western blot - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 15 seconds; Lane 2 : 3 minutes; Lane 3 : 10 seconds; Lane 4 : 15 seconds.

The MW observed is consistent with the literature : PMID19136966; PMID 23618806.

All lanes:

Western blot - Anti-EpCAM antibody [EPR20532-222] (ab213500) at 1/2000 dilution

Lane 1:

T-47D (Human ductal breast epithelial tumor cell line) whole cell lysate at 20 µg

Lane 2:

SK-OV-3 (Human ovarian cancer cell line) whole cell lysate at 20 µg

Lane 3:

HT-29 (Human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 4:

HCT 116 (Human colorectal carcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa,40 kDa

false

• WB

Lab

Western blot - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Lanes 1 - 3 : Merged signal (red and green). Green - ab213500 observed at 40 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab213500 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 ab213500 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.

All lanes:

Western blot - Anti-EpCAM antibody [EPR20532-222] (ab213500) at 1/1000 dilution

Lane 1:

Wild-type A431 whole cell lysate at 20 µg

Lane 2:

EPCAM knockout A431 whole cell lysate at 20 µg

Lane 2:

Western blot - Human EPCAM knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-epcam-knockout-a-431-cell-line-ab261902'>ab261902</a>)

Lane 3:

HeLa whole cell lysate at 20 µg

Predicted band size: 35 kDa

Observed band size: 35 kDa

false

• WB

Supplier Data

Western blot - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2 : 3 minutes; Lane 3 : 10 seconds; Lane 4 : 15 seconds.

All lanes:

Western blot - Anti-EpCAM antibody [EPR20532-222] (ab213500) at 1/2000 dilution

Lane 1:

Human breast cancer lysate at 20 µg

Lane 2:

4T1 (Mouse mammary gland carcinoma cell line) whole cell lysate at 20 µg

Lane 3:

Human colon lysate at 20 µg

Lane 4:

Mouse small intestine lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa,40 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-EpCAM antibody [EPR20532-222] (AB213500)

EpCAM was immunoprecipitated from 0.35 mg of HCT 116 (Human colorectal carcinoma cell line) whole cell lysate with ab213500 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab213500 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Lane 1 : HCT 116 whole cell lysate 10 μg (Input).

Lane 2 : ab213500 IP in HCT 116 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab213500 in HCT 116 whole cell lysate.

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-EpCAM antibody [EPR20532-222] (ab213500)

Predicted band size: 35 kDa

false

• WB

Lab

Western blot - Anti-EpCAM antibody [EPR20532-222] (AB213500)

False colour image of Western blot : Anti-EpCAM antibody [EPR20532-222] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab213500 was shown to bind specifically to EpCAM. A band was observed at 37/45 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in EPCAM knockout cell line ab281596 (knockout cell lysate ab282948). To generate this image, wild-type and EPCAM knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-EpCAM antibody [EPR20532-222] (ab213500) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

EPCAM knockout HCT 116 cell lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Lane 5:

HeLa cell lysate at 20 µg

Predicted band size: 35 kDa

Observed band size: 37-45 kDa

false

• WB

Supplier Data

Western blot - Anti-EpCAM antibody [EPR20532-222] (AB213500)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-EpCAM antibody [EPR20532-222] (ab213500) at 1/1000 dilution

All lanes:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa,40 kDa

false

Exposure time: 3min