Anti-E Cadherin antibody [4A2]

• WB

Lab

Western blot - Anti-E Cadherin antibody [4A2] (AB231303)

Western blot : Anti-CDH1 antibody [4A2] (ab231303) staining at 1 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab231303 was shown to bind specifically to CDH1. A band was observed at 130, 110, 55 kDa in wild-type A431 cell lysates with no signal observed at this size in CDH1 knockout cell line ab273747 (knockout cell lysate ab273781). To generate this image, wild-type and CDH1 knockout A431 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-E Cadherin antibody [4A2] (ab231303) at 1 µg/mL

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

Western blot - Human CDH1 (E Cadherin) knockout A-431 cell lysate (ab273781)

Lane 2:

Western blot - Human CDH1 (E Cadherin) knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-cdh1-e-cadherin-knockout-a-431-cell-line-ab273747'>ab273747</a>)

Lane 2:

CDH1 knockout A431 cell lysate at 20 µg

Lane 3:

Caco-2 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Predicted band size: 97 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [4A2] (AB231303)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon carcinoma labelling E cadherin with ab231303 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab231303 anti-E Cadherin antibody [4A2] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [4A2] (AB231303)

Immunofluorescence staining of E-Cadherin using ab231303 in wild-type A431 cells (top panel) and CDH1 knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton-X-100 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 ab231303 at 1.0 µg/mL and ab6046 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150084) (shown in red), both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a confocal section is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [4A2] (AB231303)

ab231303 staining E-Cadherin in MCF7 cells. The cells were fixed with 4% PFA (10min), 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 overnight at +4°C with ab231303 at 1μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [4A2] (AB231303)

IHC image of E Cadherin staining in a section of formalin-fixed paraffin-embedded normal human colon carcinoma* performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab231303, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [4A2] (AB231303)

Immunofluorescence staining of E-Cadherin using ab231303 in wild-type A431 cells (top panel) and CDH1 knockout A431 cells (bottom panel). The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton-X-100 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 ab231303 at 1.0 µg/mL and ab6046 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150084) (shown in red), both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a confocal section is shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [4A2] (AB231303)

IHC image of E Cadherin staining in a section of formalin-fixed paraffin-embedded normal rat large intestine performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab231303, 5ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [4A2] (AB231303)

IHC image of E-Cadherin staining in a section of formalin-fixed paraffin-embedded normal mouse large intestine performed on a Leica Biosystems BOND® RX instrument. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab231303, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• WB

Lab

Western blot - Anti-E Cadherin antibody [4A2] (AB231303)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before ab231303 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at a 1ug/ml concentration and 1/10000 dilution respectively. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-E Cadherin antibody [4A2] (ab231303)

Lane 1:

MCF7 whole cell lysate at 20 µg

Lane 2:

Mouse colon tissue lysate at 20 µg

Lane 3:

Rat colon tissue lysate at 20 µg

Predicted band size: 97 kDa

Observed band size: 105 kDa

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• WB

CiteAb

Western blot - Anti-E Cadherin antibody [4A2] (AB231303)

E Cadherin western blot using anti-E Cadherin antibody [4A2] ab231303. Publication image and figure legend from Zhu, M. X., Wei, C. Y., et al., 2019, J Exp Clin Cancer Res, PubMed 31533816.

ab231303 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab231303 please see the product overview.

TRIP13 activates AKT/mTOR pathway by interacting with ACTN4. a ACTN4 was identified as a binding partner of TRIP13 by combining Co-IP and MS. b The AKT/mTOR pathway was affected by ACTN4 interference. c Co-IP was used to validate the interaction of TRIP13 and ACTN4. d Immunofluorescence used to verify the colocalization of TRIP13 and ACTN4. e-f qRT-PCR and western blot analysis of TRIP13 or ACTN4 mRNA expression in HCC cells transfected with shTRIP13 or siACTN4. g-h Cell migration and invasion were measured in the indicated cells. i EMT markers and reprehensive genes of AKT/mTOR pathway were measured in the indicated cells by western blot. *p < 0.05, **p < 0.01, ***p < 0.001

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