All lanes : Anti-N Cadherin antibody [EPR22397-264] (ab245117) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CDH2 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 100 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab245117 observed at 125 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab245117 was shown to react with N Cadherin in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab255377 (knockout cell lysate 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 1000 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.

 

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.

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

Predicted band size: 100 kDa
Observed band size: 110,130 kDa why is the actual band size different from the predicted?

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

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Rat brain lysate
Lane 4 : Rat heart lysate
Lane 5 : Rat liver lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 100 kDa
Observed band size: 110,130 kDa why is the actual band size different from the predicted?

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).

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).