Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling VEGFA with purified ab52917 at 1:100 dilution (2.96 µg/ml).

Heat mediated antigen retrieval was performed using citrate buffer, pH 6.0.

Negative control shown in inset.

All lanes : Anti-VEGFA antibody [EP1176Y] - C-terminal (ab52917) at 1 µg/ml (unpurified)

Lane 1 : Recombinant Human VEGFA protein (His tag) (ab204773)
Lane 2 : Recombinant mouse VEGFA protein (Active) (ab185265)

Lysates/proteins at 0.1 µg per lane.

Secondary
All lanes : Rabbit IgG secondary antibody at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 27 kDa
Observed band size: 23 kDa

why is the actual band size different from the predicted?


Exposure time: 10 seconds

This blot was produced using a 10% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab52917 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

Purified ab52917 staining VEGF in NIH/3T3 (Mouse embryo fibroblast cell line) cells.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with purified ab52917 at 5 μg/ml and ab195884, at 1/250 dilution, overnight at +4°C, followed by a further incubation at room temperature for 1h with an Goat anti-Rabbit Alexa Fluor^®488 secondary (ab150081) at 2 μg/ml (shown in green).

Nuclear DNA was labeled in blue with DAPI.

Overlay histogram showing NIH/3T3 (Mouse embryo fibroblast cell line) cells stained with unpurified ab52917 (red line).

The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab52917, 1/1000 dilution) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 0.1 μg/1x10⁶ cells used under the same conditions.

Unlabeled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This antibody gave a positive signal in NIH/3T3 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used at 1/100 dilution.

Anti-VEGFA antibody [EP1176Y] - C-terminal (ab52917) at 1/10000 dilution (Purified) + His-tagged Human VEGFA (aa 27-232) recombinant protein at 0.015 µg with 5% NFDM/TBST

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 27 kDa
Observed band size: 27 kDa

Unpurified ab52917 staining VEGF in mouse kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

Tissue was fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 5% serum for 45 minutes at 25°C. Samples were incubated with primary antibody (1/400 in 4% BSA + 5% serum in PBST) for 14 hours at 4°C. An Alexa Fluor^® 546-conjugated Donkey anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

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Flow Cytometry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling VEGFA with purified ab52917 at 1:30 dilution (10 µg/ml) (red).

Cells were fixed with 4% paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black).

Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).