Anti-VAV2 antibody [EP1067Y] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VAV2 antibody [EP1067Y] - BSA and Azide free (AB239846)

Human cervical carcinoma stained with ab52640 at 1/50 - 1/100 dilution

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52640).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-VAV2 antibody [EP1067Y] - BSA and Azide free (AB239846)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling VAV2 (red) with ab52640 at a 1/2500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52640).

• IP

Unknown

Immunoprecipitation - Anti-VAV2 antibody [EP1067Y] - BSA and Azide free (AB239846)

VAV2 was immunoprecipitated using 0.5mg Hek293 whole cell extract, 10µg of Rabbit monoclonal to VAV2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hek293 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab52640.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 100kDa; VAV2.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52640).

All lanes:

Immunoprecipitation - Anti-VAV2 antibody [EP1067Y] (<a href='/en-us/products/primary-antibodies/vav2-antibody-ep1067y-ab52640'>ab52640</a>)

Predicted band size: 101 kDa

false

• WB

Lab

Western blot - Anti-VAV2 antibody [EP1067Y] - BSA and Azide free (AB239846)

This data was developed using the same antibody clone in a different buffer formulation (ab52640).

Lanes 1 - 4 : Merged signal (red and green). Green - ab52640 observed at 101 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab52640 was shown to specifically react with VAV2 in wild-type HAP1 cells as signal was lost in VAV2 knockout cells. Wild-type and VAV2 knockout samples were subjected to SDS-PAGE. ab52640 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/20000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-VAV2 antibody [EP1067Y] - BSA and Azide free (ab239846) at 1/20000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

VAV2 knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 101 kDa

false

• WB

Lab

Western blot - Anti-VAV2 antibody [EP1067Y] - BSA and Azide free (AB239846)

This data was developed using the same antibody clone in a different buffer formulation (ab52640).

Lanes 1- 2 : Merged signal (red and green). Green - ab52640 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab52640 was shown to react with VAV2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265318 (knockout cell lysate ab257794) was used. Wild-type HeLa and VAV2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab52640 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 20000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-VAV2 antibody [EP1067Y] (<a href='/en-us/products/primary-antibodies/vav2-antibody-ep1067y-ab52640'>ab52640</a>) at 1/20000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

VAV2 knockout HeLa cell lysate at 20 µg

Predicted band size: 101 kDa

Observed band size: 100 kDa

false