重组Anti-N Cadherin抗体[EPR19654] (ab207608)

All lanes : Anti-N Cadherin antibody [EPR19654] (ab207608) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : cdh2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

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

Performed under reducing conditions.

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

False colour image of Western blot: Anti-N Cadherin antibody [EPR19654] 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, ab207608 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.

All lanes : Anti-N Cadherin antibody [EPR19654] (ab207608) at 1/1000 dilution

Lane 1 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 2 : PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate
Lane 3 : Human fetal brain lysate
Lane 4 : Human cerebellum lysate
Lane 5 : Human fetal liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lanes 3-5 : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG 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 time: Lane 1/2: 30 seconds; Lane 3: 5 seconds; Lane 4/5: 3 minutes.

The molecular weight observed is consistent with what has been described in the literature(PMID: 22553038).

Immunohistochemical analysis of paraffin-embedded human cardiac muscle tissue labeling N Cadherin with ab207608 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Intercalated disc staining on human cardiac muscle is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

All lanes : ab76011, Anti-N Cadherin antibody [EPR1791-4] (Left) or ab207608 (Right) at 1/1000 dilution

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

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?

Exposure time:

Lane 1 and 2: 4 seconds
Lane 4 to 11: 1 seconds

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)

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

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling N Cadherin with ab207608 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membrane staining on human endometrium is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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