Anti-EpCAM antibody [EPR20533-63]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20533-63] (AB221552)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling EpCAM with ab221552 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. 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) Ready to use.

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

• Flow Cyt

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Flow Cytometry - Anti-EpCAM antibody [EPR20533-63] (AB221552)

Flow cytometric analysis of 4% paraformaldehyde-fixed 4T1 (mouse mammary gland carcinoma cell line) and NIH/3T3 (mouse embyro fibroblast cell line) cells labeling EpCAM with ab221552 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Flow cytometry was performed with fresh cells without fixation and permeabilization.

Negative control : NIH/3T3 (PMID : 23264216).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20533-63] (AB221552)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized 4T1 (mouse mammary gland carcinoma cell line) and NIH/3T3 (mouse embyro fibroblast cell line) cells labeling EpCAM with ab221552 at 1/100 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 4T1 cells.

Negative control : NIH/3T3 (PMID : 23264216).

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.

• Flow Cyt

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Flow Cytometry - Anti-EpCAM antibody [EPR20533-63] (AB221552)

Flow cytometry overlay histogram showing left M158 positive cells and right negative NIH3T3 cells stained with ab221552 (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 (ab221552) (1x10⁶ in 100 μL at 0.1 μg/ml) for 30 min on ice.

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

Isotype control antibody (black line) was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20533-63] (AB221552)

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling EpCAM with ab221552 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on mouse lung 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.

• IP

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Immunoprecipitation - Anti-EpCAM antibody [EPR20533-63] (AB221552)

EpCAM was immunoprecipitated from 0.35 mg of 4T1 (mouse mammary gland carcinoma cell line) whole cell lysate with ab221552 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab221552 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : 4T1 (Mouse mammary gland carcinoma) whole cell lysate 10 μg (Input).

Lane 2 : ab221552 IP in 4T1 whole cell lysate .

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab221552 in 4T1 whole cell lysate .

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

Exposure time : 30 seconds.

The band between 12-15kDa has been documented in literature as the cleaved form of EpCAM (PMID : 23409978).

All lanes:

Immunoprecipitation - Anti-EpCAM antibody [EPR20533-63] (ab221552)

Predicted band size: 35 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-EpCAM antibody [EPR20533-63] (AB221552)

EpCAM was immunoprecipitated from 0.35 mg of mouse kidney lysate with ab221552 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab221552 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Mouse kidney lysate 10 μg (Input).

Lane 2 : ab221552 IP in mouse kidney lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab221552 in mouse kidney lysate.

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

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-EpCAM antibody [EPR20533-63] (ab221552)

Predicted band size: 35 kDa

false

• WB

Supplier Data

Western blot - Anti-EpCAM antibody [EPR20533-63] (AB221552)

Blocking/Dilution buffer : 5% NFDM/TBST.

The MW observed is consistent with the literature (PMID 23618806).

The band between 12-15kDa has been documented in literature as the cleaved form of EpCAM (PMID : 23409978).

Negative control : NIH/3T3 (PMID : 23264216).

All lanes:

Western blot - Anti-EpCAM antibody [EPR20533-63] (ab221552) at 1/5000 dilution

Lane 1:

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

Lane 2:

NIH/3T3 (mouse embyro fibroblast 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: 12 kDa,39 kDa

true

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-EpCAM antibody [EPR20533-63] (AB221552)

Blocking/Dilution buffer : 5% NFDM/TBST.

The MW observed is consistent with the literature (PMID 23409978; PMID 23618806).

Negative control : NIH/3T3 (PMID : 23264216).

All lanes:

Western blot - Anti-EpCAM antibody [EPR20533-63] (ab221552) at 1/5000 dilution

Lane 1:

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

Lane 2:

NIH/3T3 (mouse embyro fibroblast 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: 39 kDa

true

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-EpCAM antibody [EPR20533-63] (AB221552)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

The MW observed is consistent with the literature (PMID 23409978; PMID 23618806).

All lanes:

Western blot - Anti-EpCAM antibody [EPR20533-63] (ab221552) at 1/5000 dilution

Lane 1:

Mouse colon lysate at 20 µg

Lane 2:

Mouse kidney lysate at 20 µg

Lane 3:

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: 39 kDa

true