Recombinant Anti-VAV3 antibody [EP1130Y] - BSA and Azide free (ab247340)

False colour image of Western blot: Anti-VAV3 antibody [EP1130Y] 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, ab52938 was shown to bind specifically to VAV3. A band was observed at 98 kDa in wild-type HeLa cell lysates with no signal observed at this size in VAV3 knockout cell line ab265419 (knockout cell lysate ab258280). To generate this image, wild-type and VAV3 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 5 % 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

This data was developed using ab52938, the same antibody clone in a different buffer formulation.

Immunocytochemistry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling VAV3 with purified ab52938 at 1/50 dilution (7.2 µg/mL). Cells were fixed in 100% Methanol and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using ab52938, the same antibody clone in a different buffer formulation.
Purified ab52938 at 1/30 dilution (2µg) immunoprecipitating VAV3 in Jurkat whole cell lysate.
Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10µg.
Lane 2 (+): ab52938 + Jurkat whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52938 in Jurkat whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.

This data was developed using ab52938, the same antibody clone in a different buffer formulation. Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labelling VAV3 with purified ab52938 at 1/40 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).