Anti-VEGF Receptor 3 antibody

• WB

Lab

Western blot - Anti-VEGF Receptor 3 antibody (AB27278)

Anti-FLT4 antibody (ab27278) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab27278 was shown to bind specifically to FLT4 but with several non-specific bands. We recommend ab243232 for Western blot. 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:

Western blot - Anti-VEGF Receptor 3 antibody (ab27278) at 1/1000 dilution

Lane 1:

HEL 92.1.7 cell lysate at 20 µg

Lane 2:

MCF7 cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 146 kDa

Observed band size: 200 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-VEGF Receptor 3 antibody (AB27278)

ICC/IF image of ab27278 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab27278, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• WB

Unknown

Western blot - Anti-VEGF Receptor 3 antibody (AB27278)

Hey cell lysate (50µg/lane) was used. The antibody was diluted at 1 : 50 for 2 hours at room tepterature Hey cell lysate (50µg/lane) was used. The antibody was diluted at 1 : 50 for 2 hours at room temperature. The protein is heavily glycosylated and this might explain the increase in MW above the predcted MW as well as the multiple bands.

All lanes:

Western blot - Anti-VEGF Receptor 3 antibody (ab27278) at 1/50 dilution

Predicted band size: 146 kDa

Observed band size: 170 kDa,200 kDa

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