HA750087

STAT3 Recombinant Rabbit Monoclonal Antibody [SY34-01]

cat.: HA750087

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, FC
Clonality:	Monoclonal
Clone number:	SY34-01

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*PBS (pH7.4).
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 88 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within N-terminal human STAT3.
Positive control:	HeLa (Human cervical adenocarcinoma cell) cell lysate, A549 (Human lung adenocarcinoma cell) cell lysate, K-562 (Human chronic myelogenous leukemia cell) cell lysate, HEK-293 (Human embryonic kidney cell) cell lysate, Hep G2 (Human hepatocellular carcinoma cell) cell lysate, PC-12 (Rat pheochromocytoma cell (undifferentiated)) cell lysate, NIH/3T3 (Mouse fibroblast) cell lysate, HeLa, NIH/3T3, human stomach carcinoma tissue, human breast carcinoma tissue, mouse brain tissue.
Subcellular location:	Cytoplasm, Nucleus.
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P FC	1:1,000-1:2,000 1:100 1:100-1:500 1:50-1:1,000 1:1,000
Uniprot #:	SwissProt: P40763 Human \| P42227 Mouse \| P52631 Rat
Alternative names:	1110034C02Rik Acute Phase Response Factor Acute-phase response factor ADMIO APRF AW109958 DNA binding protein APRF FLJ20882 HIES MGC16063 Signal transducer and activator of transcription 3 (acute phase response factor) Signal transducer and activator of transcription 3 STAT 3 Stat3 STAT3_HUMAN

Images

Fig1: Western blot analysis of STAT3 on different lysates with Rabbit anti-STAT3 antibody (HA750087) at 1/1,000 dilution.

Lane 1: HeLa (Human cervical adenocarcinoma cell) cell lysate
Lane 2: A549 (Human lung adenocarcinoma cell) cell lysate
Lane 3: K-562 (Human chronic myelogenous leukemia cell) cell lysate
Lane 4: HEK-293 (Human embryonic kidney cell) cell lysate
Lane 5: Hep G2 (Human hepatocellular carcinoma cell) cell lysate
Lane 6: PC-12 (Rat pheochromocytoma cell (undifferentiated)) cell lysate
Lane 7: NIH/3T3 (Mouse fibroblast) cell lysate

Lysates/proteins at 20 µg/Lane.
Exposure time: 3 minutes; ECL: K1801

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA750087, 1/1,000 in 5% NFDM/TBST, overnight at 4 ℃
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

Predicted band size: 88 kDa
Observed band size: 88 kDa

Fig2: Western blot analysis of STAT3 on different lysates with Rabbit anti-STAT3 antibody (HA750087) at 1/500 dilution.

Lane 1: Hela-si NT cell lysate
Lane 2: Hela-si STAT3 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 88 kDa
Observed band size: 88 kDa

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

ET1605-45 was shown to specifically react with STAT3 in Hela-si NT cells. No band was observed when Hela-si STAT3 sample was tested. Hela-si NT and Hela-si STAT3 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 (ET1609-76, 1/500) and Loading control antibody (Rabbit anti-Hsp90, ET1605-56, 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.

Fig3: Immunocytochemistry analysis of HeLa cells labeling STAT3 with Rabbit anti-STAT3 antibody (HA750087) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-STAT3 antibody (HA750087) at 1/100 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 (HA601187, 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.

Fig4: Immunocytochemistry analysis of NIH/3T3 cells labeling STAT3 with Rabbit anti-STAT3 antibody (HA750087) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-STAT3 antibody (HA750087) at 1/100 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 (HA601187, 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.

	Fig5: Immunohistochemical analysis of paraffin-embedded human stomach carcinoma tissue using anti-STAT3 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750087, 1/50) for 30 minutes 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.
	Fig6: Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-STAT3 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750087, 1/50) for 30 minutes 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.
	Fig7: Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-STAT3 antibody (HA750087) at 1/1,000 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 (HA750087) at 1/1,000 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.
	Fig8: Flow cytometric analysis of HeLa cells labeling STAT3. Cells were fixed and permeabilized. Then stained with the primary antibody (HA750087, 1/1,000) (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).

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.