Anti-GATA3 抗体 [EPR16651] - ChIP Grade

• WB

Unknown

Western blot - Anti-GATA3 antibody [EPR16651] - ChIP Grade (AB199428)

Exposure

Lane1 : 26 seconds

Lane 2 : 15 seconds

All lanes:

Western blot - Anti-GATA3 antibody [EPR16651] - ChIP Grade (ab199428) 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='/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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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA3 antibody [EPR16651] - ChIP Grade (AB199428)

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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA3 antibody [EPR16651] - ChIP Grade (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).

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-GATA3 antibody [EPR16651] - ChIP Grade (AB199428)

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 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-GATA3 antibody [EPR16651] - ChIP Grade (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).

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GATA3 antibody [EPR16651] - ChIP Grade (AB199428)

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.

• ChIP

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ChIP - Anti-GATA3 antibody [EPR16651] - ChIP Grade (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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA3 antibody [EPR16651] - ChIP Grade (AB199428)

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] - ChIP Grade (AB199428)

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

Supplier Data

Western blot - Anti-GATA3 antibody [EPR16651] - ChIP Grade (AB199428)

Blocking/dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GATA3 antibody [EPR16651] - ChIP Grade (ab199428) at 1/1000 dilution

All lanes:

SH-SY5Y (Human neuroblastoma from bone marrow cells) cell extract at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa,62 kDa

Observed band size: 48 kDa

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Exposure time: 1min

• WB

Lab

Western blot - Anti-GATA3 antibody [EPR16651] - ChIP Grade (AB199428)

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.

All lanes:

Western blot - Anti-GATA3 antibody [EPR16651] - ChIP Grade (ab199428) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

GATA3 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

Jurkat whole cell lysate at 20 µg

Predicted band size: 47 kDa

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• WB

Lab

Western blot - Anti-GATA3 antibody [EPR16651] - ChIP Grade (AB199428)

Western blot : Rabbit Monoclonal[EPR16651] to GATA3 ab199428 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 50 kDa in Wild-type A549 cell lysates with no signal observed at this size in GATA3 knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-GATA3 antibody [EPR16651] - ChIP Grade (ab199428) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human GATA3 knockout A549 cell line (<a href='/products/cell-lines/human-gata3-knockout-a549-cell-line-ab286669'>ab286669</a>) at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 48 kDa

Observed band size: 50 kDa

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