All lanes : Anti-Vimentin antibody [V9] - Cytoskeleton Marker (ab8069) at 1 µg

Lane 1 : U-2 OS cell lysate
Lane 2 : Human tonsil cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : VIM knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 54 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab8069).

Lanes 1 - 4: Merged signal (red and green). Green - ab8069 observed at 53 kDa. Red - loading control, ab181602 observed at 37 kDa.

 ab8069 was shown to react with Vimentin in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255446 (knockout cell lysate ab263775) was used. Wild-type and Vimentin knockout samples were subjected to SDS-PAGE. ab8069 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 µg/ml and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

 

This image was produced using the same antibody clone, but in a different formulation, ab8069.

ab8069 staining Vimentin (colored green) in wild-type HAP1 cells (top panel) and VIM knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab8069 at 0.5μg/ml and ab202272 (Rabbit monoclonal [EP1332Y] to alpha Tubulin (Alexa Fluor^® 594) at 1/250 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with ab150117 (Goat secondary antibody to Mouse IgG (Alexa Fluor^® 488) at 2 μg/ml (colored green). Nuclear DNA was labeled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This image was produced using the same antibody clone, but in a different formulation, ab8069.

IHC image of ab8069 staining Vimentin in normal human kidney formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8069, 1/500 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX.

No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Flow cytometry overlay histogram showing wild-type HAP1 (green line) and VIM knockout HAP1 cells stained with ab223871 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized 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 (ab223871) (1x10⁶ in 100 µl at 0.04 µg/ml) for 30 min at 22°C.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody was mouse IgG1&kappa (ab170190) used at the same concentration and conditions as the primary antibody (wild-type HAP1 - black line VIM knockout HAP1 - 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.

This antibody can also be used in in HAP1 cells fixed with 4% formaldehyde (10 min) / permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

 

All lanes : Anti-Vimentin antibody [V9] - Cytoskeleton Marker (ab8069) at 1 µg/ml

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : VIM (Vimentin) knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MOLT4 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 54 kDa

This image was produced using the same antibody clone, but in a different formulation, ab8069.

Lanes 1 - 4: Merged signal (red and green). Green - ab8069 observed at 57 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab8069 was shown to specifically react with Vimentin in wild-type HAP1 cells as signal was lost in VIM (Vimentin) knockout cells. Wild-type and VIM (Vimentin) knockout samples were subjected to SDS-PAGE. Ab8069 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/10000 dilution respectively. Blots were developed with goat anti-mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and goat anti-rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

This image was produced using the same antibody clone, but in a different formulation, ab8069.

ab8069 staining Vimentin in HeLa cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab8069 at 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 2µg/ml (shown in red). Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This image was produced using the same antibody clone, but in a different formulation, ab8069.

IHC image of Vimentin staining in human Hodgkin's lymphoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8069, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX.

This image was produced using the same antibody clone, but in a different formulation, ab8069.

IHC image showing no staining when ab8069 was used on mouse kidney formalin fixed paraffin embedded tissue, using MOM detection kit, ab127055. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30 mins. The section was incubated with ab8069 at 5 µg/ml for 15 mins at room temperature. DAB was used as the chromogen. The section was counterstained with hematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This image was produced using the same antibody clone, but in a different formulation, ab8069.

Overlay histogram showing SV40LT-SMC cells stained with ab8069 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab8069, 0.1μg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor^® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 0.1μg/1x10⁶ cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This antibody gave a positive signal in SV40LT-SMC cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 20 min used under the same conditions.

This image was produced using the same antibody clone, but in a different formulation, ab8069.

Overlay histogram showing MDA-MB-231 cells stained with ab8069 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab8069, 1μg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor^® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x10⁶ cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This antibody gave a positive signal in MDA-MB-231 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 20 min used under the same conditions.