重组Anti-GATA2 + GATA3抗体[EPR24359-3] (ab272237)

Primary Antibodies used:

ab272237 Anti-GATA2+GATA3 antibody 1:1000 dilution (0.4 μg/ml)

ab109241 Anti-GATA2 antibody 1:1000 dilution (0.3μg/ml)

ab199428 Anti-GATA3 antibody 1:1000 dilution (0.6 μg/ml)

These tests were run in parallel on the same lysates to show the expected expression pattern for GATA3 and GATA2 respectively.

K-562 express high levels of GATA2 but no expression for GATA3 (PMID: 23636060).

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

This blot was developed using a higher sensitivity ECL substrate.

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID: 21666600).

Exposure time: 81 seconds.

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

Negative control: U-937 (PMID: 19212333).

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

Exposure time: 125 seconds.

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

Negative control: DU 145 (PMID: 33586682 ); Daudi (PMID: 19212333); Jurkat (PMID: 19212333).

Exposure time: 125 seconds.

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labelling GATA2 + GATA3 with ab272237 at 1/500 (0.858 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on human placenta (PMID: 28232602).The section was incubated with ab272237 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human prostate cancer tissue labelling GATA2+ GATA3 with ab272237 at 1/500 (0.858 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on human prostate cancer (PMID: 31296150). The section was incubated with ab272237 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized U-937 (Human histiocytic lymphoma monocyte, Left)/ HeLa (Human cervix adenocarcinoma epithelial cell, Right) cells labelling GATA2+ GATA3 with ab272237 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Negative control: U-937 cell (PMID: 19212333).

GATA2+ GATA3 was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab272237 at 1/30 dilution (2 ug in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab272237 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 ug

Lane 2: ab272237 IP in K-562 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab272237 in K-562 whole cell lysate

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

Exposure time: 76 seconds.

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labelling GATA2 + GATA3 with ab272237 at 1/500 (0.858 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on the distal tubules and collecting ducts of mouse kidney (PMID: 18202227). The section was incubated with ab272237 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling GATA2 + GATA3 with ab272237 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labelling GATA2 + GATA3 with ab272237 at 1/500 (0.858 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on the distal tubules and collecting ducts of rat kidney. The section was incubated with ab272237 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.