Immunocytochemistry/ Immunofluorescence - Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168)
ab33168 staining VE Cadherin in HUV-EC cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab33168 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
Western blot - Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168)
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.
All lanes:
Western blot - Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168) at 1 µg/mL
All lanes:
HUVEC Cell Lysate at 10 µg
Secondary
All lanes:
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: 88 kDa
Observed band size: 120 kDa
Exposure time: 1min
Western blot - Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168)
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.
All lanes:
Western blot - Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168) at 1 µg/mL
All lanes:
HUVEC Cell Lysate at 10 µg
Secondary
All lanes:
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: 88 kDa
Observed band size: 120 kDa, 55 kDa
Exposure time: 1min
Western blot - Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168)
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.
All lanes:
Western blot - Anti-VE Cadherin antibody - Intercellular Junction Marker (ab33168) at 1 µg/mL
All lanes:
HUVEC Cell Lysate at 10 µg
Secondary
All lanes:
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: 88 kDa
Observed band size: 115 kDa, 117 kDa, 45 kDa
Exposure time: 1min
