STAT3 pTyr705 (703-714) Mouse Monoclonal Antibody [Clone ID: PS3/1]

This antibody reacts with human and mouse STAT3 phosphorylated at Tyr705, and zebrafish STAT3 phosphorylated at Tyr708.

Store undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer.
Avoid repeated freezing and thawing.

STAT (Signal Transducers and Activators of Transcription) proteins play important roles in development, cell differentiation, and cell cycle control. STAT3 is an ~85 kDa protein involved in the signaling pathways of many cytokines and growth factors, including GCSF and IL-6, where it functions as a negative regulator of transcription. STAT3 is also constitutively activated in a number of human tumors and it possesses anti-apoptotic activity and oncogenic potential. STAT3 may also regulate apoptosis by inhibiting NF?B. Activation of STAT3 by tyrosine phosphorylation results in dimerization, nuclear translocation, and DNA binding.

SDS-PAGE & Western Blotting
1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) containing appropriate protease inhibitors. Incubate it at 4 oC with rotating for 30 minutes, then sonicate briefly (up to 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 oC and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the Lysis buffer to make 8 mg/mL solution.
3) Mix the sample with equal volume of Laemmli’s sample buffer.
4) Boil the samples for 2 minutes and centrifuge. Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis.
5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system. (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure.
6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 oC.
7) Incubate the membrane with the anti-phospho-zebrafish STAT3 (Tyr708) monoclonal antibody (1 µg/mL) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
8) Wash the membrane with PBS (5 minutes x 6 times).
9) Incubate the membrane with the 1:10000 POD-conjugated anti-mouse IgG diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
10) Wash the membrane with PBS (5 minutes x 6 times).
11) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap.
12) Expose to X-ray film in a dark room for 5 minutes. Develop the film as usual. The conditions for exposure and development may vary.