Anti-ZEB1 antibody [EPR17375]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZEB1 antibody [EPR17375] (AB203829)

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling ZEB1 with ab203829 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on Human breast carcinoma tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ZEB1 antibody [EPR17375] (AB203829)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling ZEB1 with ab203829 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab203829 at 1/250 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-ZEB1 antibody [EPR17375] (AB203829)

Intracellular Flow Cytometry analysis of HeLa cells labelling ZEB1 with ab203829 at 1/1000 (red). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. An Alexa Fluorr^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZEB1 antibody [EPR17375] (AB203829)

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling ZEB1 with ab203829 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on Human cervix carcinoma tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-ZEB1 antibody [EPR17375] (AB203829)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ZEB1 antibody [EPR17375] (ab203829) at 1/500 dilution

Lane 1:

MDA-MB231 （Human breast adenocarcinoma epithelial cell）whole cell lysates at 20 µg

Lane 2:

SKOV3 (Human ovarian cancer epithelial cell) whole cell lysates at 20 µg

Lane 3:

MCF7 （Human breast adenocarcinoma epithelial cell）whole cell lysates at 20 µg

Lane 4:

A549 （Human lung carcinoma epithelial cell） whole cell lysates at 20 µg

Lane 5:

Human Ovary lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 124 kDa

Observed band size: 200 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-ZEB1 antibody [EPR17375] (AB203829)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : ZEB1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab203829 observed at 200 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab203829 was shown to specifically react with ZEB1 in wild-type HAP1 cells as signal was lost in ZEB1 knockout cells. Wild-type and ZEB1 knockout samples were subjected to SDS-PAGE. ab203829 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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.

Lane 1:

Western blot - Anti-ZEB1 antibody [EPR17375] (ab203829)

Lanes 2 - 3:

Western blot - Anti-ZEB1 antibody [EPR17375] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/zeb1-antibody-epr17375-bsa-and-azide-free-ab228986'>ab228986</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

ZEB1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Predicted band size: 124 kDa

false

• WB

Supplier Data

Western blot - Anti-ZEB1 antibody [EPR17375] (AB203829)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ZEB1 antibody [EPR17375] (ab203829) at 1/1000 dilution

All lanes:

HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 124 kDa

Observed band size: 200 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-ZEB1 antibody [EPR17375] (AB203829)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ZEB1 antibody [EPR17375] (ab203829) at 1/1000 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 124 kDa

Observed band size: 200 kDa

false

Exposure time: 30s