Chromatin Immunoprecipitation (ChIP) was performed using commercial Protein-G coated 96 well high-throughput ChIP assay kit by loading 3 µg of cross-linked chromatin samples from HeLa cells starved overnight and then treated with IL-6 with either A) 1:50 dilution of Go-ChIP-Grade™ Purified anti-STAT3 Phospho (Tyr705) (Clone 13A3-1), B) equal amount of Purified Mouse IgG1, κ Isotype Control Antibody (#400101), or C) competitor’s ChIP-grade Purified anti-STAT3 Phospho (Tyr705) Antibody and D) equal amount of matched Isotype Control Antibody as recommended by the manufacturer. The enriched DNA was purified and quantified by real-time qPCR using primers targeting human IRF1 gene region. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the 5% of total amount of input chromatin.
Chromatin Immunoprecipitation (ChIP) was performed using commercial Protein-G coated 96 well high-throughput ChIP assay kit by loading 3 µg of cross-linked chromatin samples from HeLa cells starved overnight and then treated with IL-6 with either A) 1:50 dilution of Go-ChIP-Grade™ Purified anti-STAT3 Phospho (Tyr705) (Clone 13A3-1), B) equal amount of Purified Mouse IgG1, κ Isotype Control Antibody (#400101), or C) competitor’s ChIP-grade Purified anti-STAT3 Phospho (Tyr705) Antibody and D) equal amount of matched Isotype Control Antibody as recommended by the manufacturer. The enriched DNA was purified and quantified by real-time qPCR using primers targeting human IRF1 gene region. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the 5% of total amount of input chromatin.
Total cell lysate from PC3 (lane 1, 15 µg), untreated HepG2 (lane 2, 15 µg), HepG2 treated with 10 ng/ml of IL-6 for 15 minutes (lane 3, 15 µg) and HeLa (lane 4, 15 µg) were resolved by electrophoresis (4-12% Bis-Tris), transferred to nitrocellulose, and probed with 1 µg/ml Purified anti-STAT3 Phospho (Tyr705) antibody (clone 13A3-1). Proteins were visualized using an HRP Goat anti-mouse IgG (clone Poly4053) antibody and chemiluminescence detection. Direct-Blot™ HRP anti-β-actin antibody (clone 2F1-1) was used as a loading control.
15 µg of total cell lysate from untreated NIH3T3 (lane 1) and NIH3T3 treated with 100 ng/ml of IL-6 for 30 minutes (lane 2) were resolved by electrophoresis (4-20% Tris-Glycine), transferred to nitrocellulose, and probed with 1 µg/ml purified anti-STAT3 Phospho (Tyr705) antibody (clone 13A3-1). Proteins were visualized using an HRP Goat anti-mouse IgG (clone Poly4053) antibody and chemiluminescence detection. Direct-Blot™ HRP anti-β-actin antibody (clone 2F1-1) was used as a loading control.
This product is scheduled for discontinuation. Please use the availability checker to determine current availability. Cat. Nos. 651001, 651002 are available as alternatives.
Description
Tyrosine phosphorylation of STAT3 at Tyr705 occurs in response to LIF, IL-6, leptin, OSM, EGF, PDGF, and HGF. It plays a key role in cell growth and apoptosis through mediating expression of a variety of genes in response to the stimuli.
Each lot of this antibody is quality control tested by ChIP Assay. The suggested dilution for ChIP application is 1:50 by volume. For Western blotting the suggested working dilution is 0.5 µg per ml antibody dilution buffer for each mini-gel.
It is recommended that the reagent to be titrated for optimal performance before each experiment.
Application Notes
The STAT3 Phospho (Tyr705) antibody recognizes the regulatory tyrosine phosphorylation of human STAT3 protein and has been shown to be useful for Western blotting.
STAT3 is a 770 amino acid protein of 88 kD. It consists of a DNA binding domain, a SH2 domain, a regulatory tyrosine responsible for binding of SH2 domain, and a C-terminal transactivation domain.
Distribution
Ubiquitous.
Function
STAT3 is tyrosine phosphorylated by receptor kinases in response to a variety of cytokines and growth factors. It forms homo- or heterodimer with STAT1 when tyrosine is phosphorylated, and then translocates to nucleus, acting as a transcription regulator. It is also essential for the differentiation of TH17 cells, which is involved in autoimmune diseases.
Cell Sources
Cytoplasm. Translocate to nucleus in response to tyrosine phosphorylation.
This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
If you need assistance with selecting the best format contact our expert technical support team.
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