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[Bio101] Western Blot for Detecting Phosphorylated STAT3
[Bio101] 蛋白印记法检测STAT3磷酸化水平   

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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.

Materials and Reagents

  1. Tumor cell lines with constitutive activation of STAT3 (positive control).
    1. DU145 (ATCC, catalog number: HTB-81 ™)
    2. HepG2 (ATCC, catalog number: HB-8065 ™)
    3. Hep3B (ATCC, catalog number: HB-8064 ™)
    4. Huh7
  2. Phospho-Stat3 (Tyr705) (D3A7) XP™ Rabbit (Cell Signaling Technology, catalog number: 9145 )
  3. Stat3 (124H6) Mouse mAb (Cell Signaling Technology, catalog number: 9139 )
  4. HRP Goat Anti-Rabbit I (BD Biosciences, catalog number: 554021 )
  5. β-Actin (13E5) Rabbit mAb (Cell Signaling Technology, catalog number: 4970 )
  6. 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.
  7. Phosphate buffered saline (PBS)
  8. 1x halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, catalog number: 78440 )
  9. M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, catalog number: 78501 )
  10. Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
  11. 10x Tris/Glycine/SDS (Bio-Rad Laboratories, catalog number: 161-0771 )
  12. Methanol (Thermo Fisher Scientific, catalog number: A412-20 )
  13. Tris buffered saline (Bio-Rad Laboratories, catalog number: 170-6435 )
  14. Tween-20 (Santa Cruz Biotechnology, catalog number: sc-29113 )
  15. Bovine serum albumin (BSA) (MP Biomedicals, catalog number: 810033 )
  16. Supersignal west Dura extended duration substrate (Thermo Fisher Scientific, catalog number: 34075 )
  17. Precision plus protein dual color standards (Bio-Rad Laboratories, catalog number: 161-0374 )
  18. Restore plus western blot stripping buffer (Thermo Fisher Scientific, catalog number: 46430 )
  19. Protein lysis buffer (see Recipes)
  20. Electrophoresis buffer (see Recipes)
  21. 1x Tris buffered saline (TBS) (see Recipes)
  22. Transfer buffer (see Recipes)
  23. Blocking buffer (see Recipes)
  24. Wash buffer (see Recipes)
  25. Primary antibody dilution buffer (see Recipes)
  26. Blotting membrane (see Recipes)

Equipment

  1. Microcentrifuges (Eppendorf, model: 5415 R )
  2. Thermolyne Rotomix (BioSurplus, model: 50800 )
  3. Microcentrifuge tubes
  4. Nitrocellulose or PVDF membrane
  5. SmartSpec plus spectrophotometer (Bio-Rad Laboratories)

Procedure

  1. Protein blotting
    1. Treat cells by adding fresh media containing regulator for desired time.
    2. Aspirate media from cultures; wash cells with 1x PBS; aspirate.
    3. Lyse cells by adding 1x protein lysis buffer.
    4. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. 
    5. Incubate at room temperature (RT) for 5 min.
    6. Microcentrifuge for 10 min at 13,000 rpm.
    7. Transfer supernatant to a clean microcentrifuge tube.
    8. Measure the protein concentration using the Bio-Rad protein assay dye reagent.
    9. Heat a 20 μg sample to 95-100 °C for 10 min; cool on ice.
    10. Load 20 μg sample onto SDS-PAGE gel (10 cm x 10 cm) and load 7 μl precision plus protein fual color standards to determine molecular weights.
    11. Electrophoresis at constant 80 Volts until the protein dye reaches the bottom of the gel.
    12. Electrotransfer to nitrocellulose or PVDF membrane using transfer buffer (constant 30 Volts overnight at 4 °C).

  2. Membrane blocking and detection of pSTAT3
    1. Incubate membrane in 25 ml of blocking buffer for 1 h at RT.
    2. Wash three times for 5 min each with 15 ml of TBS/T.
    3. Incubate membrane and primary phospho-Stat3 (Tyr705) (D3A7) XP™ rabbit antibody (1:1,000) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4 °C.
    4. Wash three times for 5 min each with 15 ml of TBS/T.
    5. Incubate membrane with HRP-conjugated goat anti-rabbit secondary antibody (1:5,000) and HRP-conjugated anti-biotin antibody (1:1,000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 h at RT.
    6. Wash three times for 5 min each with 15 ml of TBS/T.
    7. Incubate membrane with 4 ml Supersignal west dura extended duration substrate with gentle agitation for 5 min at RT.
    8. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and expose to x-ray film. An initial 10 sec exposure should indicate the proper exposure time.

  3. Detection of STAT3
    1. Wash the membrane three times for 5 min each with 15 ml of TBS/T.
    2. Incubate membrane with 10 ml restore plus western blot stripping buffer for 15 min to stripe the bonding antibodies.
    3. Wash the membrane three times for 5 min each with 15 ml of TBS/T.
    4. Repeat steps 11-18 and use Stat3 (124H6) mouse mAb as primary antibody and HRP-conjugated anti-mouse IgG as secondary antibody.

  4. Detection of β-actin
    1. Repeat steps 19-22 for β-actin detection.

  5. Analyzing the level of STAT3 activation
    1. Scan the film and measure the intensity of pSTAT3 and STAT3 bands. Calculate the STAT3 activation using the following formula:
      Percentage of STAT3 activation = (intensity of pSTAT3)/ (intensity of STAT3 + intensity of pSTAT3) x 100%.

Recipes

  1. Electrophoresis buffer
    To prepare 1 L buffer, add 100 ml 10x Tris/glycine to 900 ml ddH2O.
  2. Protein lysis buffer
    M-PER mammalian protein extraction reagent with 1x halt protease and phosphatase inhibitor cocktail.
  3. Transfer buffer
    To prepare 1 L buffer, add 100 ml 10x Tris/Glycine/SDS and 200 ml methanol to 700 ml ddH2O.
  4. 1x TBS
    To prepare 1 L of 1x TBS, add 100 ml 10x TBS to 900 ml ddH2O.
  5. Blocking buffer
    1x TBS, 0.1% tween-20 with 5% (w/v) BSA.
  6. Wash buffer (TBS/T)
    1x TBS, 0.1% tween-20
  7. Primary antibody dilution buffer
    1x TBS, 0.1% tween-20 with 5% BSA
  8. Blotting membrane
    This protocol has been optimized for nitrocellulose membranes, which CST recommends. PVDF membranes may also be used.

References

  1. Campbell, C. L., Jiang, Z., Savarese, D. M. and Savarese, T. M. (2001). Increased expression of the interleukin-11 receptor and evidence of STAT3 activation in prostate carcinoma. Am J Pathol 158(1): 25-32.
  2. Fujimoto, M., Naka, T., Nakagawa, R., Kawazoe, Y., Morita, Y., Tateishi, A., Okumura, K., Narazaki, M. and Kishimoto, T. (2000). Defective thymocyte development and perturbed homeostasis of T cells in STAT-induced STAT inhibitor-1/suppressors of cytokine signaling-1 transgenic mice. J Immunol 165(4): 1799-1806.

简介

STAT3转录因子是许多细胞因子和生长因子受体的重要信号分子,并且在许多人肿瘤中被组成型激活,并且具有致癌潜力和抗凋亡活性。 STAT3被Tyr705处的磷酸化激活,其诱导二聚化,核转位和DNA结合。 蛋白质印迹最常用于通过使用对磷酸化酪氨酸特异的抗体来检测STAT3的活化。

材料和试剂

  1. 具有STAT3的组成型激活的肿瘤细胞系(阳性对照)。
    1. DU145(ATCC,目录号:HTB-81 TM)
    2. HepG2(ATCC,目录号:HB-8065 TM)
    3. Hep3B(ATCC,目录号:HB-8064 TM)
    4. Huh7
  2. Phospho-Stat3(Tyr705)(D3A7)XP TM兔(Cell Signaling Technology,目录号:9145)
  3. Stat3(124H6)小鼠mAb(Cell Signaling Technology,目录号:9139)
  4. HRP山羊抗兔I(BD Biosciences,目录号:554021)
  5. β-肌动蛋白(13E5)兔mAb(Cell Signaling Technology,目录号:4970)
  6. 抗小鼠IgG,HRP-连接的抗体(Cell Signaling Technology,目录号:7076)
    注意:上述抗体已经被作者测试,可能被用户需要的抗体替代。
  7. 磷酸盐缓冲盐水(PBS)
  8. 1x halt蛋白酶和磷酸酶抑制剂混合物(Thermo Fisher Scientific,目录号:78440)
  9. M-PER哺乳动物蛋白提取试剂(Thermo Fisher Scientific,目录号:78501)
  10. Bio-Rad蛋白测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:500-0006)
  11. 10x Tris /甘氨酸/SDS(Bio-Rad Laboratories,目录号:161-0771)
  12. 甲醇(Thermo Fisher Scientific,目录号:A412-20)
  13. Tris缓冲盐水(Bio-Rad Laboratories,目录号:170-6435)
  14. Tween-20(Santa Cruz Biotechnology,目录号:sc-29113)
  15. 牛血清白蛋白(BSA)(MP Biomedicals,目录号:810033)
  16. Supersignal West Dura延长期限底物(Thermo Fisher Scientific,目录号:34075)
  17. 精密加蛋白双色标准品(Bio-Rad Laboratories,目录号:161-0374)
  18. 还原+ western印迹剥离缓冲液(Thermo Fisher Scientific,目录号:46430)
  19. 蛋白裂解缓冲液(见配方)
  20. 电泳缓冲液(见配方)
  21. 1×Tris缓冲盐水(TBS)(见Recipes)
  22. 转移缓冲液(见配方)
  23. 阻塞缓冲区(参见配方)
  24. 洗涤缓冲液(见配方)
  25. 一级抗体稀释缓冲液(见配方)
  26. 印迹膜(见配方)

设备

  1. 微量离心机(Eppendorf,型号:5415R)
  2. Thermolyne Rotomix(BioSurplus,型号:50800)
  3. 微量离心管
  4. 硝化纤维素或PVDF膜
  5. SmartSpec plus分光光度计(Bio-Rad Laboratories)

程序

  1. 蛋白质印迹
    1. 通过添加含有调节剂的新鲜培养基所需时间处理细胞。
    2. 从培养物中吸出培养基; 用1x PBS洗涤细胞; 吸出。
    3. 通过加入1×蛋白裂解缓冲液裂解细胞。
    4. 立即从板上刮下细胞,并将提取物转移到微量离心管中。
    5. 在室温(RT)孵育5分钟。
    6. 在13,000rpm微量离心10分钟。
    7. 将上清转移到干净的微量离心管中。
    8. 使用Bio-Rad蛋白测定染料试剂测量蛋白质浓度。
    9. 将20μg样品加热至95-100℃10分钟; 在冰上冷却。
    10. 加载20微克样品在SDS-PAGE凝胶(10厘米×10厘米)和负载7微升精度加蛋白质色标准,以确定分子量。
    11. 在恒定的80伏电泳,直到蛋白质染料到达凝胶的底部。
    12. 使用转移缓冲液(在4℃恒定30伏过夜)电转移至硝酸纤维素或PVDF膜
  2. 膜阻断和检测pSTAT3
    1. 孵育膜在25毫升封闭缓冲液在室温下1小时。
    2. 用15ml TBS/T洗涤三次,每次5分钟。
    3. 孵育膜和初始磷酸Stat3(Tyr705)(D3A7)XP™兔抗体(1:1,000)在10ml一抗稀释缓冲液中,轻轻搅拌在4℃过夜。
    4. 用15ml TBS/T洗涤三次,每次5分钟。
    5. 与HRP缀合的山羊抗兔二抗(1:5,000)和HRP-缀合的抗生物素抗体(1:1,000)孵育膜,以检测生物素化蛋白质标记在10ml封闭缓冲液,温和搅拌1小时在室温。
    6. 用15ml TBS/T洗涤三次,每次5分钟。
    7. 孵育膜与4毫升Supersignal西硬膜扩展持续时间基板与温和搅拌5分钟在室温。
    8. 排出过量显影溶液的膜(不要让其干燥),用塑料包装包裹并暴露于X射线胶片。 初始10秒暴露应指示适当的暴露时间。

  3. STAT3的检测
    1. 用15ml TBS/T洗膜三次,每次5分钟。
    2. 孵育膜与10毫升恢复+蛋白质印迹剥离缓冲液15分钟条纹的绑定抗体。
    3. 用15ml TBS/T洗膜三次,每次5分钟。
    4. 重复步骤11-18,并使用Stat3(124H6)小鼠mAb作为第一抗体和HRP缀合的抗小鼠IgG作为第二抗体。

  4. β-肌动蛋白的检测
    1. 重复步骤19-22以检测β-肌动蛋白。

  5. 分析STAT3激活的水平
    1. 扫描膜并测量pSTAT3和STAT3条带的强度。 使用以下公式计算STAT3激活:
      STAT3活化百分比=(pSTAT3的强度)/(STAT3的强度+ pSTAT3的强度)×100%。

食谱

  1. 电泳缓冲液
    为了制备1L缓冲液,将100ml 10×Tris /甘氨酸加入到900ml ddH 2 O中。
  2. 蛋白裂解缓冲液
    具有1x停止蛋白酶和磷酸酶抑制剂混合物的M-PER哺乳动物蛋白质提取试剂
  3. 传输缓冲区
    为了制备1L缓冲液,将100ml 10×Tris /甘氨酸/SDS和200ml甲醇加入到700ml ddH 2 O中。
  4. 1x TBS
    为了制备1L的1×TBS,将100ml 10×TBS加入900ml ddH 2 O中。
  5. 阻塞缓冲区
    1×TBS,0.1%Tween-20和5%(w/v)BSA
  6. 洗涤缓冲液(TBS/T)
    1×TBS,0.1%tween-20
  7. 一级抗体稀释缓冲液
    1×TBS,0.1%Tween-20和5%BSA
  8. 印迹膜
    该协议已针对硝酸纤维素膜进行了优化,CST建议。 也可以使用PVDF膜

参考文献

  1. Campbell,C.L.,Jiang,Z.,Savarese,D.M。和Savarese,T.M。(2001)。 白细胞介素-11受体的表达增加和前列腺癌中STAT3活化的证据。 Am J Pathol 158(1):25-32。
  2. Fujimoto,M.,Naka,T.,Nakagawa,R.,Kawazoe,Y.,Morita,Y.,Tateishi,A.,Okumura,K.,Narazaki,M.and Kishimoto,T。(2000)。 STAT诱导的STAT抑制剂-1 /细胞因子抑制因子中T细胞的胸腺细胞发育和扰乱的体内平衡缺陷 信号转导-1转基因小鼠。 J Immunol 165(4):1799-1806。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, H. (2011). Western Blot for Detecting Phosphorylated STAT3. Bio-protocol Bio101: e125. DOI: 10.21769/BioProtoc.125;
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Yuanqing Lin
8/31/2011 4:08:20 PM Reply
bio-protocol

过夜我一般是从晚上6点到第二天早上8点,时间长一点也没关系。
蛋白质印迹第三步由于已经加了蛋白裂解液,细胞已经被裂解了。贴壁的细胞用胰酶消化后离心,再用裂解液重悬细胞沉淀也可以,大多数时候我就是这样做的。如果对时间点要求比较高的话,我一般是把所有的不同时间点的细胞沉淀先收集冻在-80度,然后一起裂解分离蛋白。

8/31/2011 4:09:04 PM