Anti-N Cadherin antibody [EPR22397-264]

• Flow Cyt

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Flow Cytometry - Anti-N Cadherin antibody [EPR22397-264] (AB245117)

Flow cytometric analysis of MCF7 (human breast adenocarcinoma epithelial cell line, Left) / HeLa (human cervix adenocarcinoma epithelial cell line, Right) cell lines labeling N Cadherin with ab245117 at 1/500 (red) compared with a Rabbit monoclonal IgG (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.

Negative control : MCF7 (PMID : 9177902).

Gated on viable cells.

• IP

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Immunoprecipitation - Anti-N Cadherin antibody [EPR22397-264] (AB245117)

N Cadherin was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell line) whole cell lysate with ab245117 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab245117 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (Input).
Lane 2 : ab245117 IP in HeLa whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab245117 in HeLa whole cell lysate.

Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 3 seconds.

The molecular weight is consistent with literature (PMID : 8230319).

All lanes:

Immunoprecipitation - Anti-N Cadherin antibody [EPR22397-264] (ab245117)

Predicted band size: 99 kDa

Observed band size: 110 kDa,130 kDa

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• WB

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Western blot - Anti-N Cadherin antibody [EPR22397-264] (AB245117)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lanes 1-3 : 26 seconds; Lanes 4-6 : 3 minutes; Lane 7 : 48 seconds.

The molecular weight is consistent with literature (PMID : 8230319).

Negative control : MCF7 (PMID : 9177902).

All lanes:

Western blot - Anti-N Cadherin antibody [EPR22397-264] (ab245117) at 1/1000 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 4:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

C6 (rat glial tumor glial cell) whole cell lysate at 10 µg

Lane 6:

A549 (human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 7:

Human brain 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: 99 kDa

Observed band size: 110 kDa,130 kDa

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• WB

Lab

Western blot - Anti-N Cadherin antibody [EPR22397-264] (AB245117)

Lanes 1-2 : Merged signal (red and green). Green - ab245117 observed at 110, 130 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab245117 was shown to react with N Cadherin in wild-type HEK-293T cells in Western blot. Loss of signal was observed when knockout sample ab263843 was used. Wild-type and N Cadherin knockout samples were subjected to SDS-PAGE. ab245117 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 100000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-N Cadherin antibody [EPR22397-264] (ab245117) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

CDH2 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human CDH2 (N Cadherin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-cdh2-n-cadherin-knockout-hek-293t-cell-line-ab255377'>ab255377</a>)

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 99 kDa

Observed band size: 125 kDa,130 kDa

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• WB

Lab

Western blot - Anti-N Cadherin antibody [EPR22397-264] (AB245117)

False colour image of Western blot : Anti-N Cadherin antibody [EPR22397-264] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab245117 was shown to bind specifically to N Cadherin. A band was observed at 125 kDa in wild-type HeLa cell lysates with no signal observed at this size in cdh2 knockout cell line ab274934 (knockout cell lysate ab274992). To generate this image, wild-type and cdh2 knockout HeLa 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.

Lanes 1 - 2:

Western blot - Anti-N Cadherin antibody [EPR22397-264] (ab245117) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-N Cadherin antibody [EPR22397-264] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/n-cadherin-antibody-epr22397-264-bsa-and-azide-free-ab245827'>ab245827</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CDH2 (N cadherin) knockout HeLa cell line (ab274934)

Lane 2:

Western blot - Human CDH2 (N Cadherin) knockout HeLa cell lysate (ab274992)

Lane 2:

cdh2 knockout HeLa cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 125 kDa

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• WB

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Western blot - Anti-N Cadherin antibody [EPR22397-264] (AB245117)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lane 1 : 3 minutes; Lane 2 : 26 seconds; Lane 3 : 48 seconds; Lane 4 : 10 seconds; Lane 5 : 3 minutes.

The molecular weight is consistent with literature (PMID : 8230319).

All lanes:

Western blot - Anti-N Cadherin antibody [EPR22397-264] (ab245117) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Rat brain lysate at 10 µg

Lane 4:

Rat heart lysate at 10 µg

Lane 5:

Rat liver lysate at 10 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: 99 kDa

Observed band size: 110 kDa,130 kDa

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