Anti-Vimentin antibody [SP20], prediluted

• Flow Cyt

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Flow Cytometry - Anti-Vimentin antibody [SP20], prediluted (AB27608)

Overlay histogram showing HeLa cells stained with ab27608 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 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 (ab27608, neat) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). 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 HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vimentin antibody [SP20], prediluted (AB27608)

Human melanoma stained with prediluted ab27608.

• WB

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Western blot - Anti-Vimentin antibody [SP20], prediluted (AB27608)

This image refers to the non-prediluted version of this product (ab16900)

This image was generated using a previous batch manufactured using the hybridoma production method.

Lane 1:

Anti-MDM2 antibody [4B11] (<a href='/en-us/products/unavailable/mdm2-antibody-4b11-ab16900'>ab16900</a>) at 1/100 dilution

Lane 1:

Western blot - Anti-Vimentin antibody [SP20] (<a href='/en-us/products/primary-antibodies/vimentin-antibody-sp20-ab16700'>ab16700</a>) at 1/100 dilution

All lanes:

HeLa cell lysate

Predicted band size: 53 kDa

Observed band size: 53 kDa

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• WB

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Western blot - Anti-Vimentin antibody [SP20], prediluted (AB27608)

This image was generated using a previous batch manufactured using the hybridoma production method.

This data was developed using the same antibody clone in a different buffer formulation (ab16700).

Lanes 1 - 4 : Merged signal (red and green). Green - ab16700 observed at 53 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab16700 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. ab16700 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 100 dilution and 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.

Lanes 1 - 4:

Western blot - Anti-Vimentin antibody [SP20], prediluted (ab27608) at 1/100 dilution

Lanes 1 - 4:

Western blot - Anti-Vimentin antibody [SP20] (<a href='/en-us/products/primary-antibodies/vimentin-antibody-sp20-ab16700'>ab16700</a>) at 1/100 dilution

Lane 1:

U20S cell lysate at 20 µg

Lane 2:

Human tonsil cell lysate at 20 µg

Lane 2:

Western blot - Human VIM (Vimentin) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-vim-vimentin-knockout-hela-cell-line-ab255446'>ab255446</a>)

Lane 3:

Wild-type HeLa cell lysate at 20 µg

Lane 4:

VIM knockout HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 53 kDa

Observed band size: 37 kDa,53 kDa

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