ET1607-39

Phospho-STAT3 (S727) Recombinant Rabbit Monoclonal Antibody [SY24-09]

cat.: ET1607-39

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Rat, Mouse
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, IP
Clonality:	Monoclonal
Clone number:	SY24-09

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ . Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	88 kDa
Isotype:	IgG
Immunogen:	Synthetic phospho-peptide corresponding to residues surrounding Ser727 of Human STAT3 aa 701-750 / 770.
Positive control:	Hela cell lysate, A431 cell lysate, NIH/3T3 cell lysate, Jurkat cell lysate, rat liver tissue, rat brain tissue, rat kidney tissue, human lung tissue, rat hippocampus tissue, human kidney tissue.
Subcellular location:	Cytoplasm, Nucleus.
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P IP	1:500-1:2,000 1:50-1:200 1:50-1:200 1:500-1:1,000 Use at an assay dependent concentration.
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 Phospho-STAT3(S727) on NIH-3T3 cell lysates.

Lane 1: NIH-3T3 cells, whole cell lysates, 10 μg/lane.
Lane 2: NIH-3T3 cells were starved 6h, whole cell lysates, 10 μg/lane.
Lane 3/4: NIH-3T3 cells were starved 6h, then treated with 200 ng/ml EGF for 10 min, whole cell lysates, 10 μg/lane.
Lane 5: NIH-3T3 cells were starved 6h, then treated with 200 ng/ml EGF for 10 min, and then treated with 2.8μg/ul lambda-PP for 30 minutes, whole cell lysates, 10 μg/lane.

Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody Anti-Phospho-STAT3(S727)(ET1607-39, 1/500) , Anti-STAT3 antibody ( ET1607-38, 1/500) and Anti-GAPDH antibody (ET1601-4, 1/10,000)was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

Predicted band size: 88 kDa
Observed band size: 88 kDa
Exposure time: Lane 1/2/3 1 minute 28 seconds
Lane 4/5 30 seconds

Fig2: All lanes: Western blot analysis of Phospho-STAT3(S727) with anti-Phospho-STAT3(S727) antibody [SY24-09] (ET1607-39) at 1:500 dilution.

Lane 1: Wild-type CT26.wt whole cell lysate.
Lane 2: STAT3 knockdown CT26.wt whole cell lysate.

ET1607-39 was shown to specifically react with Phospho-STAT3(S727) in wild-type CT26.wt cells. Weakened band was observed when STAT3 knockdown samples were tested. Wild-type and STAT3 knockdown 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 Anti-Phospho-STAT3(S727) antibody (ET1607-39, 1/500) and Anti-HSP90 antibody (ET1605-56, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co., Ltd., Guangzhou, China).

	Fig3: Western blot analysis of Phospho-STAT3(S727) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1607-39, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature. Positive control: Lane 1: Hela cell lysate Lane 2: A431 cell lysate Lane 3: NIH/3T3 cell lysate Lane 4: Jurkat cell lysate
	Fig4: ICC staining of Phospho-STAT3 (S727) in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1607-39, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
	Fig5: ICC staining of Phospho-STAT3 (S727) in A431 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1607-39, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
	Fig6: ICC staining of Phospho-STAT3 (S727) in NIH/3T3 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1607-39, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

	Fig7: Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1:500 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1607-39) at 1:500 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: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1:500 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1607-39) at 1:500 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.
	Fig9: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1:500 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1607-39) at 1:500 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.

Fig10: Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (ET1607-39) 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.

Fig11: Immunohistochemical analysis of paraffin-embedded mouse liver tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (ET1607-39) 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.

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