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- Analytical tools
Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (Sepharose® Bead Conjugate) #4074
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.Learn more about how we get our images
Gallery: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (Sepharose® Bead Conjugate) #4074
Immunoprecipitation for Analysis by Western Blotting
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
- 20X Phosphate Buffered Saline (PBS): (#9808).
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*.
- 3X SDS Sample Buffer: (#7722) 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue
- 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix.
- ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.
B. Preparing Cell Lysates
- Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
- To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
- Remove PBS and add 0.5 ml 1X ice-cold cell lysis buffer to each plate (10 cm) and incubate the plates on ice for 5 minutes.
- Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
- Sonicate samples on ice three times for 5 seconds each.
- Microcentrifuge for 10 minutes at 4°C, 14,000 x g, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.
- Take 200 μl cell lysate and add 10 μl of the immobilized antibody, incubate with rotation overnight at 4°C.
- Microcentrifuge for 30 seconds at 4°C. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice during washes.
- Proceed to sample analysis by western blotting or kinase activity (section D).
D. Sample Analysis
Proceed to one of the following specific set of steps.
For Analysis by Western Immunoblotting
- Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
- Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
- Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
- Analyze sample by western blot (see Western Immunoblotting Protocol).
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).
For Analysis by Kinase Assay
- Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
- Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
- Incubate for 30 min at 30°C.
- Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
- Transfer supernatant containing phosphorylated substrate to another tube.
- Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
- Load the sample (15–30 µl) on SDS-PAGE (4–20%).
posted December 2007
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, Monkey Species 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.
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).
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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc.