Anti-VAV3 antibody [EP1130Y]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-VAV3 antibody [EP1130Y] (AB52938)

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).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-VAV3 antibody [EP1130Y] (AB52938)

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.

• IP

Lab

Immunoprecipitation - Anti-VAV3 antibody [EP1130Y] (AB52938)

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.

All lanes:

Immunoprecipitation - Anti-VAV3 antibody [EP1130Y] (ab52938)

Predicted band size: 98 kDa

Observed band size: 98 kDa

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• WB

Lab

Western blot - Anti-VAV3 antibody [EP1130Y] (AB52938)

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.

Lanes 1 and 3:

Western blot - Anti-VAV3 antibody [EP1130Y] (ab52938) at 1/1000 dilution

Lane 2:

Western blot - Anti-VAV3 antibody [EP1130Y] (ab52938)

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Empty

Lane 2:

Western blot - Human VAV3 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-vav3-knockout-hela-cell-line-ab265419'>ab265419</a>)

Lane 3:

VAV3 knockout HeLa cell lysate at 20 µg

Predicted band size: 98 kDa

Observed band size: 98 kDa

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• WB

Unknown

Western blot - Anti-VAV3 antibody [EP1130Y] (AB52938)

All lanes:

Western blot - Anti-VAV3 antibody [EP1130Y] (ab52938) at 1/1000 dilution

All lanes:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 98 kDa

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