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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous 79, 86 Rabbit IgG
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Immunoprecipitation

Immunoprecipitation of NIH/3T3 cell lysates using Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423 and Stat3 (79D7) Rabbit mAb (Sepharose® Bead Conjugate). The western blot was probed using Stat3 (124H6) Mouse mAb #9139.

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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.

  1. 20X Phosphate Buffered Saline (PBS): (#9808).
  2. 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*.

  1. 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
  2. 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.
  3. 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

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
  3. 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.
  4. Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate samples on ice three times for 5 seconds each.
  6. 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.

C. Immunoprecipitation

  1. Take 200 μl cell lysate and add 10 μl of the immobilized antibody, incubate with rotation overnight at 4°C.
  2. Microcentrifuge for 30 seconds at 4°C. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice during washes.
  3. 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

  1. Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
  4. 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

  1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15–30 µl) on SDS-PAGE (4–20%).

posted December 2007

protocol id: 27

Product Usage Information

Application Dilutions
Immunoprecipitation 1:20

Storage: 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.

Specificity / Sensitivity

Stat3 (79D7) Rabbit mAb (Sepharose® Bead Conjugate) detects endogenous levels of total Stat3 protein.


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a Stat3 fusion protein corresponding to the carboxy-terminal sequence of mouse Stat3 protein.

Product Description

This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. Stat3 (79D7) Rabbit mAb (Sepharose® Bead Conjugate) is useful for the immunoprecipitation assay of Stat3 proteins.


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).


1.  Heim, M.H. (2001) J Recept Signal Transduct Res 19, 75-120.

2.  Takeda, K. et al. (1997) Proc Natl Acad Sci U S A 94, 3801-4.

3.  Catlett-Falcone, R. et al. (1999) Immunity 10, 105-15.

4.  Garcia, R. and Jove, R. (1998) J Biomed Sci 5, 79-85.

5.  Bromberg, J.F. et al. (1999) Cell 98, 295-303.

6.  Darnell, J.E. et al. (1994) Science 264, 1415-21.

7.  Ihle, J.N. (1995) Nature 377, 591-4.

8.  Wen, Z. et al. (1995) Cell 82, 241-50.

9.  Yokogami, K. et al. (2000) Curr Biol 10, 47-50.

10.  Biethahn, S. et al. (1999) Exp Hematol 27, 885-94.


Entrez-Gene Id 6774
Swiss-Prot Acc. P40763


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 5,675,063.