Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168) at 1 µg/ml + HUVEC Cell Lysate at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 87 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab33168 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

 

The band we observe at 115 kDa is believed to be the glycosylated form of the protein.

Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168) at 1 µg/ml + HUVEC Cell Lysate at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 87 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?
Additional bands at: 55 kDa (possible non-specific binding)


Exposure time: 1 minute

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab33168 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

 

The band we observe at 115 kDa is believed to be the glycosylated form of the protein.

ICC/IF image of VE-Cadherin staining on HUVEC cells using ab33168. The cells were incubated with the primary antibody (ab33168) and the secondary was FITC conjugated anti-rabbit used at 1:400. The cells were incubated with only the secondary antibody as a negative control.

ab33168 stained in HUVEC cells. Cells were fixed with 100% methanol (5min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab33168 at 1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature

This image was generated using confluent cells.

ICC/IF image of VE Cadherin stained HUVEC cells. The cells were incubated with the antibody ab33168 at 1/150 (Green). The cells were also stained with Rhodamine phalloidin (Red).

Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168) at 1 µg/ml + HUVEC Cell Lysate at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 87 kDa
Observed band size: 115,117 kDa why is the actual band size different from the predicted?
Additional bands at: 45 kDa (possible non-specific binding)


Exposure time: 1 minute

The observed band for Cadherin 5 has a higher molecular weight of 115kDa due to glycosylation of the protein. 

The immunogen used to raise this antibody has 89% homology with Cadherin 18, 88kDa , which we believe is the additional observed band at 117kDa, again due to glycosylation of the protein. 

ab33168 staining VE Cadherin in the endothelial cell line from Human liver by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with Paraformaldehyde. Samples were incubated with primary antibody (1/100 in PBS + 2.5% BSA + 0.1% triton) for 1 hour at 37°C. Alexa Fluor 594 Chicken anti-Rabbit IgG (H+L) Cross-Adsorbed Secon was used as the secondary antibody at 4 µg/ml.

See Abreview