Anti-Vinculin antibody [EPR8185](AB129002)

ab129002 (purified) at 1/20 immunoprecipitating vinculin in HeLa cells. Lane 1: HeLa whole cell lysate (10 μg). Lane 2: HeLa whole cell lysate (10 μg). Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab129002 in HeLa whole cell lysate. For western blotting, a HRP-conjugated goat anti-rabbit antibody was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes: Immunoprecipitation - Anti-Vinculin antibody [EPR8185] (ab129002)

Predicted band size: 124 kDa

Immunofluorescent staining of HEK293 cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab129002 at a dilution of 1/50. An Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) was used as the secondary at a dilution of 1/1000 and the cells were counter stained with DAPI. The negative controls are shown in the bottom middle and right hand panels. For negative control 1, the primary was used and then goat anti-mouse IgG was used at a dilution of 1/500. For negative control 2, a mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) were used.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes: Western blot - Anti-Vinculin antibody [EPR8185] (ab129002) at 1/10000 dilution

Lane 1: HepG2 cell lysate at 20 µg

Lane 2: HeLa cell lysate at 20 µg

Lane 3: PC3 cell lysate at 20 µg

Secondary

All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 124 kDa

Observed band size: 124 kDa

Intracellular Flow Cytometry analysis of 293 (human embryonic kidney epithelial) cells labeling Vinculin (red) with ab129002 at a 1/200 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluorr® 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.

Different batches of ab129002 were tested on HepG2 (Human hepatocellular carcinoma epithelial cell) lysate at 1.0 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 124 kDa.

All lanes: Western blot - Anti-Vinculin antibody [EPR8185] (ab129002)

Predicted band size: 124 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes: Western blot - Anti-Vinculin antibody [EPR8185] (ab129002) at 1/20000 dilution

Lane 1: C6 cell lysate at 10 µg

Lane 2: rat kidney cell lysate at 10 µg

Secondary

All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 124 kDa

Observed band size: 124 kDa