All lanes : Anti-N Cadherin antibody [EPR1791-4] (ab76011) at 1/1000 dilution

Lane 1 : HeLa Whole Cell Lysate
Lane 2 : HeLa Whole Cell Lysate (Scraped)
Lane 3 : Human Brain Tissue Lysate
Lane 4 : Mouse Brain Tissue Lysate
Lane 5 : Rat Brain Tissue Lysate
Lane 6 : MCF7 Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 100 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?

This blot was produced using a 4-12% Bis-tris 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 blocked for an hour using 3% milk before ab76011 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/20000 dilution respectively. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cardiac muscle tissue sections labeling N Cadherin with purified ab76011 at 1:50 dilution (1.94 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-N Cadherin antibody [EPR1791-4] (ab76011) at 1/5000 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 - ab76011 observed at 125 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab76011 was shown to react with N Cadherin in wild-type HEK-293T. 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. ab76011 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 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 : ab76011, Anti-N Cadherin antibody [EPR1791-4] (Left) or ab207608, Anti-N Cadherin antibody [EPR19654] (Right) at 1/1000 dilution

Lane 1 : A549 (Human lung carcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method with 5% NFDM/TBST
Lane 2 : PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method with 5% NFDM/TBST
Lane 3 : HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method with 5% NFDM/TBST
Lane 4 : HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST
Lane 5 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate prepared in RIPA lysis method with 5% NFDM/TBST
Lane 6 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate prepared in 1%SDS Hot lysis method with 5% NFDM/TBST
Lane 7 : C6 (Rat glial tumor glial cell) whole cell lysates prepared in RIPA lysis method with 5% NFDM/TBST
Lane 8 : C6 (Rat glial tumor glial cell) whole cell lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST
Lane 9 : Human brain lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST
Lane 10 : Mouse brain lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST
Lane 11 : Rat brain lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 22553038). This antibody fails to detect N Cadherin in HCT 116 cell which is positive described in the literature (PMID: 23431386 and 26540342)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human liver tissue sections labeling N Cadherin with purified ab76011 at 1:50 dilution (1.94 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry of kidney carcinoma staining N Cadherin with ab76011 at 1μg/ml

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab76011 staining N Cadherin in Mouse pancreas tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA + 1% FBS for 2 hours at room temperature; antigen retrieval was by heat mediation in a citrate buffer pH6. Samples were incubated with primary antibody (1/500 in 1% BSA + 1% FBS) for 16 hours at 4°C. An undiluted HRP-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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