STAT6 Recombinant Rabbit Monoclonal Antibody [SY13-09]

Western blot analysis of STAT6 on different lysates with Rabbit anti-STAT6 antibody (ET1605-49) at 1/2,000 dilution.

Lane 1: THP-1 cell lysate (20 µg/Lane)
Lane 2: Jurkat cell lysate (20 µg/Lane)
Lane 3: Raji cell lysate (20 µg/Lane)
Lane 4: HeLa cell lysate (20 µg/Lane)
Lane 5: MDA-MB-231 cell lysate (20 µg/Lane)
Lane 6: NIH/3T3 cell lysate (20 µg/Lane)
Lane 7: RAW264.7 cell lysate (20 µg/Lane)

Predicted band size: 94 kDa
Observed band size: 100 kDa

Exposure time: 2 minutes;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1605-49) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

☑ Knockdown (KD)

Western blot analysis of STAT6 on different lysates with Rabbit anti-STAT6 antibody (ET1605-49) at 1/500 dilution.

Lane 1: Hela-si NT cell lysate
Lane 2: Hela-si STAT6 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 94 kDa
Observed band size: 100 kDa

Exposure time: 1 minute 34 seconds;
4-20% SDS-PAGE gel.

ET1605-49 was shown to specifically react with STAT6 in Hela-si NT cells. Weakened band was observed when Hela-si STAT6 sample was tested. Hela-si NT and Hela-si STAT6 samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1605-49, 1/500) and Loading control antibody (Rabbit anti-GAPDH, ET1601-4, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-rabbit IgG-HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.

STAT6 was immunoprecipitated from 0.5 mg Hela whole cell lysates with ET1605-49 at 2 μg/mL. Western blot was performed from the immunoprecipitate using ET1605-49 at 1/500 dilution for 45 minutes at room temperature. Goat anti-Rabbit IgG-HRP Secondary Antibody (HA1001) was used at 1:300,000 dilution for 30 minutes at room temperature.

Lane 1: Hela whole cell lysates at 10 μg;
Lane 2: STAT6 (ET1605-49) IP in Hela whole cell lysates;
Lane 3: Rabbit IgG instead of STAT6 (ET1605-49) in Hela whole cell lysates.

Predicted band size: 94 kDa
Observed band size: 100 kDa

Exposure time: 2 minutes;

8% SDS-PAGE gel.

Immunohistochemical analysis of paraffin-embedded human solitary fibrous tumor tissue with Rabbit anti-STAT6 antibody (ET1605-49) at 1/1,000 dilution.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. The section was incubated with ET1605-49 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-STAT6 antibody (ET1605-49) at 1/3,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1605-49) at 1/3,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-STAT6 antibody (ET1605-49) at 1/3,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1605-49) at 1/3,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-STAT6 antibody (ET1605-49) at 1/3,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1605-49) at 1/3,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunocytochemistry analysis of MDA-MB-231 cells labeling STAT6 with Rabbit anti-STAT6 antibody (ET1605-49) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-STAT6 antibody (ET1605-49) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Flow cytometric analysis of HeLa cells labeling STAT6.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1605-49, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Application: IF-Tissue

Species: Mouse

Site: kidney

Sample: Paraffin-embedded section

Antibody concentration: 1/1,000

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Solitary fibrous tumor
Sample: Paraffin-embedded section

Primary antibody dilution: 1/1,000
Antigen retrieval: ER2
Platform: Leica Biosystems BOND® RX