Anti-GATA3 antibody [EPR16651] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

This data was developed using ab199428, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human renal cell carcinoma labelling GATA3 with ab199428 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab199428 Anti-GATA3 antibody [EPR16651] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

Intracellular 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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

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).

• ChIP

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ChIP - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

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).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

This data was developed using ab199428, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse breast tissue labeling GATA3 with ab199428 at 1/500 dilution.

The section was incubated with ab199428 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Citrate buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

This data was developed using ab199428, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse breast cancer tissue labeling GATA3 with ab199428 at 1/500 dilution.

The section was incubated with ab199428 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Citrate buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins.

• WB

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Western blot - Anti-GATA3 antibody [EPR16651] - BSA and Azide free (AB214804)

Exposure

Lane1 : 26 seconds

Lane 2 : 15 seconds

All lanes:

Western blot - Anti-GATA3 antibody [EPR16651] - ChIP Grade (<a href='/en-us/products/primary-antibodies/gata3-antibody-epr16651-chip-grade-ab199428'>ab199428</a>) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate 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 1/100000 dilution

Predicted band size: 47 kDa

Observed band size: 48 kDa

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