Immunoprecipitation of HeLa cell lysates, untreated or treated with IFN-α, using XP® Rabbit (DA1E) IgG (Sepharose® Bead Conjugate) #3423 (lanes 1 and 2) and Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (Sepharose® Bead Conjugate) (lanes 3 and 4). The western blot was probed using Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138.
|REACTIVITY||H M R Mk|
|MW (kDa)||79, 86|
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (Sepharose® Bead Conjugate) is useful for the immunoprecipitation of phospho-Stat3 (Tyr705). This antibody was tested using human cell lysates and is expected to exhibit the same species cross-reactivity as the unconjugated antibody (Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 27
Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (Sepharose® Bead Conjugate) detects endogenous levels of Stat3 only when phosphorylated at Tyr705. This antibody does not cross-react with phospho-EGFR or the corresponding phospho-tyrosines of other Stat proteins.Species Reactivity:
Human, Mouse, Rat, MonkeySpecies predicted to react based on 100% sequence homology:
Hamster, Bovine, Pig, Horse
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr705 of mouse Stat3 protein.
The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors (1) and is required for murine fetal development (2). Research studies have shown that Stat3 is constitutively activated in a number of human tumors (3,4) and possesses oncogenic potential (5) and anti-apoptotic activities (3). Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding (6,7). Transcriptional activation seems to be regulated by phosphorylation at Ser727 through the MAPK or mTOR pathways (8,9). Stat3 isoform expression appears to reflect biological function as the relative expression levels of Stat3α (86 kDa) and Stat3β (79 kDa) depend on cell type, ligand exposure, or cell maturation stage (10). It is notable that Stat3β lacks the serine phosphorylation site within the carboxy-terminal transcriptional activation domain (8).
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