All lanes : Anti-GATA3 antibody [EPR16651] - ChIP Grade (ab199428) at 1/100 dilution

All lanes : Mouse brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 48 kDa

Exposure

Lane1: 26 seconds

Lane 2: 15 seconds

All lanes : Anti-GATA3 antibody [EPR16651] - ChIP Grade (ab199428) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : GATA3 knockout HAP1 whole cell lysate
Lane 3 : Jurkat whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 48 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab199428 observed at 48 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab199428 was shown to recognize GATA3 in wild-type HAP1 cells as signal was lost at the expected MW in GATA3 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GATA3 knockout samples were subjected to SDS-PAGE. Ab199428 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab199428).

Immunohistochemical analysis of paraffin-embedded Human neuroblastoma tissue labeling GATA3 with ab199428 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on Human neuroblastoma tissue is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary ab, secondary ab is Goat Anti-Rabbit IgG H&L (HRP) (ab97051).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab199428).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SH-SY5Y cells (Human neuroblastoma from bone marrow cells) labeling GATA3 with ab199428 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear staining on SH-SY5Y cell line is observed. The nuclear counterstain 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 1/500 dilution (red).
The negative controls are as follows:-
-ve control 1 - ab199428 at 1/250 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/500 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 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab199428).

Chromatin was prepared from MCF7 cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 2μg of ab199428 (blue), and 20μl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the IgG control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach). Primers and probes are located in the first kb of the transcribed region.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab199428).

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling GATA3 with ab199428 at 1/500 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.
Negative control: Used PBS instead of primary ab, secondary ab is Goat Anti-Rabbit IgG H&L (HRP) (ab97051).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab199428).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Clone EPR16651 (ab214804) has been successfully conjugated by Abcam. This image was generated using Anti-GATA3 antibody [EPR16651] (PE). Please refer to ab225419 for protocol details.

Overlay histogram showing MCF7 cells stained with ab225419 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab225419, 1/1000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

Flow Cytometry analysis of Jurkat cells labelling GATA3 with ab199428 at 1/500 (red). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. An Alexa Fluor^® 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab199428).

Clone EPR16651 (ab214804) has been successfully conjugated by Abcam. This image was generated using Anti-GATA3 antibody [EPR16651] (Alexa Fluor® 647). Please refer to ab208896 for protocol details.

ab208896 staining GATA3 in T47D cells. The cells were fixed with 4% formaldehyde (10 min), 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 ab208896 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

 

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

 

This product also gave a positive signal under the same testing conditions in T47D cells fixed with 100% methanol (5 min).

Clone EPR16651 (ab214804) has been successfully conjugated by Abcam. This image was generated using Anti-GATA3 antibody [EPR16651] (Alexa Fluor® 488). Please refer to ab208895 for protocol details.

ab208895 staining GATA3 in T47D cells. The cells were fixed with 4% formaldehyde (10 min), 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 ab208895 at 1/1000 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

 

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

 

This product also gave a positive signal under the same testing conditions in T47D cells fixed with 100% methanol (5 min).