Anti-Vinculin antibody [EPR8185] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

This data was developed using ab196454, the same antibody clone in a different buffer formulation.

Flow cytometry overlay histogram of 4% paraformaldehyde HEK-293 (human embryonic kidney epithelial cell) cells permeabilised with 90% methanol labelling Vinculin with ab196454 at 1/500 dilution (Red line) compared with a Rabbit monoclonal IgG (ab172730) as Isotype control (Black line) and an unlabelled control (cells without incubation with primary antibody) (Blue line).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab129002).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

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 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 (ab129002).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

Immunofluorescent staining of vinculin in HUVEC cells with unpurified ab129002 at 1/100 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab129002).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

This data was developed using ab129002, the same antibody clone in a different buffer formulation.

Flow cytometry overlay histogram showing left HeLa positive cells and right negative HeLa VCL KO stained with ab129002 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab129002) (1x 10⁶ in 100μl at 0.008μg/ml (1/7875)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab129002).

Flow cytometry overlay histogram showing wild-type A-431 (green line) and VCL knockout A-431 stained with ab129002 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab129002) (1x 106 in 100μl at 0.00032 μg/ml (1/6249999)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type A-431 - black line, VCL knockout A-431 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IP

Lab

Immunoprecipitation - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab129002).

All lanes:

Immunoprecipitation - Anti-Vinculin antibody [EPR8185] - Loading Control (<a href='/en-us/products/primary-antibodies/vinculin-antibody-epr8185-loading-control-ab129002'>ab129002</a>)

Predicted band size: 124 kDa

false

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

This data was developed using ab196454, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol fixed NIH/3T3 (mouse embryonic fibroblasts) permeabilized using 0.1% Triton X-100 labelling Vinculin with ab196454 at 1/50 dilution (green). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (magenta) was used as a counterstain. Nuclear DNA was labelled with DAPI (shown in blue). Image showing cytoplasmic staining in NIH/3T3 cell line was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

This data was developed using ab196454, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol fixed C6 (rat glial tumor glial cell) permeabilized using 0.1% Triton X-100 labelling Vinculin with ab196454 used at 1/50 dilution (green). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (magenta) was used as a counterstain. Nuclear DNA was labelled with DAPI (shown in blue). Image showing cytoplasmic staining in C6 cell line was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

This data was developed using ab196454, the same antibody clone in a different buffer formulation.

Flow cytometry overlay histogram of 4% paraformaldehyde C6 (rat glial tumor glial cell) cells permeabilised with 90% methanol labelling Vinculin with ab196454 at 1/500 dilution (Red line) compared with a Rabbit monoclonal IgG (ab172730) as Isotype control (Black line) and an unlabelled control (cells without incubation with primary antibody) (Blue line).

• WB

Lab

Western blot - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

This data was developed using ab129002, the same antibody clone in a different buffer formulation. 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] - Loading Control (<a href='/en-us/products/primary-antibodies/vinculin-antibody-epr8185-loading-control-ab129002'>ab129002</a>)

Predicted band size: 124 kDa

false

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

This data was developed using ab196454, the same antibody clone in a different buffer formulation.

Flow cytometry overlay histogram of 4% paraformaldehyde HUVEC (human umbilical vein endothelial cell) cells permeabilised with 90% methanol labelling Vinculin with ab196454 at 1/500 dilution (Red line) compared with a Rabbit monoclonal IgG (ab172730) as Isotype control (Black line) and an unlabelled control (cells without incubation with primary antibody) (Blue line).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Vinculin antibody [EPR8185] - BSA and Azide free (AB217171)

This data was developed using ab196454, the same antibody clone in a different buffer formulation.

Flow cytometry overlay histogram of 4% paraformaldehyde NIH/3T3 (mouse embryonic fibroblast) cells permeabilised with 90% methanol labelling Vinculin with ab196454 at 1/500 dilution (Red line) compared with a Rabbit monoclonal IgG (ab172730) as Isotype control (Black line) and an unlabelled control (cells without incubation with primary antibody) (Blue line).