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Western Blot for Detecting Phosphorylated STAT3
Published: Aug 20, 2011 DOI: 10.21769/BioProtoc.125 Views: 26281
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Abstract
The STAT3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors and is constitutively activated in a number of human tumors and possesses oncogenic potential and anti-apoptotic activities. STAT3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding. Western blot is most commonly used to detect the activation of STAT3 by using an antibody that is specific for the phosphorylated tyrosine705.
Background
Materials and Reagents
- Tumor cell lines with constitutive activation of STAT3 (positive control).
- Phospho-Stat3 (Tyr705) (D3A7) XP™ Rabbit (Cell Signaling Technology, catalog number: 9145 )
- Stat3 (124H6) Mouse mAb (Cell Signaling Technology, catalog number: 9139 )
- HRP Goat Anti-Rabbit I (BD Biosciences, catalog number: 554021 )
- β-Actin (13E5) Rabbit mAb (Cell Signaling Technology, catalog number: 4970 )
- Anti-mouse IgG, HRP-linked Antibody (Cell Signaling Technology, catalog number: 7076 )
Note: The above antibodies have been tested by the author and may be substituted with the antibodies desired by users. - Phosphate buffered saline (PBS)
- 1x halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, catalog number: 78440 )
- M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, catalog number: 78501 )
- Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
- 10x Tris/Glycine/SDS (Bio-Rad Laboratories, catalog number: 161-0771 )
- Methanol (Thermo Fisher Scientific, catalog number: A412-20 )
- Tris buffered saline (Bio-Rad Laboratories, catalog number: 170-6435 )
- Tween-20 (Santa Cruz Biotechnology, catalog number: sc-29113 )
- Bovine serum albumin (BSA) (MP Biomedicals, catalog number: 810033 )
- Supersignal west Dura extended duration substrate (Thermo Fisher Scientific, catalog number: 34075 )
- Precision plus protein dual color standards (Bio-Rad Laboratories, catalog number: 161-0374 )
- Restore plus western blot stripping buffer (Thermo Fisher Scientific, catalog number: 46430 )
- Protein lysis buffer (see Recipes)
- Electrophoresis buffer (see Recipes)
- 1x Tris buffered saline (TBS) (see Recipes)
- Transfer buffer (see Recipes)
- Blocking buffer (see Recipes)
- Wash buffer (see Recipes)
- Primary antibody dilution buffer (see Recipes)
- Blotting membrane (see Recipes)
Equipment
- Microcentrifuges (Eppendorf, model: 5415 R )
- Thermolyne Rotomix (BioSurplus, model: 50800 )
- Microcentrifuge tubes
- Nitrocellulose or PVDF membrane
- SmartSpec plus spectrophotometer (Bio-Rad Laboratories)
Procedure
Category
Cancer Biology > Inflammation > Biochemical assays > Protein analysis
Biochemistry > Protein > Immunodetection > Western blot
Cell Biology > Cell signaling > Phosphorylation
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