All lanes : Anti-VAV2 antibody [EP1067Y] (ab52640) at 1/20000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : VAV2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 101 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

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.

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

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

All lanes : Anti-VAV2 antibody [EP1067Y] (ab52640) at 1/20000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : VAV2 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 101 kDa

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.

Anti-VAV2 antibody [EP1067Y] (ab52640) at 1/20000 dilution + 293 cell lysate at 10 µg

Secondary
Goat anti-rabbit HRP labeled at 1/2000 dilution

Predicted band size: 101 kDa
Observed band size: 101 kDa

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.

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.