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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.

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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

癌症生物学 > 炎症 > 生物化学试验 > 蛋白质分析
作者: Huagang Zhang
Huagang ZhangAffiliation: Albert Einstein College of Medicine, Yeshiva University, New York City, USA
For correspondence: huagangzhang@gmail.com
Bio-protocol author page: a21
8/20/2011, 11896 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.125

[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.

[Abstract] Stat3转录因子是许多细胞因子和生长因子受体中重要的信号分子,它在许多人类的肿瘤细胞中持续激活,具备潜在的致癌能力和抗凋亡能力。Stat3通过705位酪氨酸磷酸化被激活,进而诱导它的二聚化,核定位以及DNA结合功能。STAT3的活性经常通过能识别705位酪氨酸磷酸化的抗体来检测。

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.

材料和试剂

 

I.  抗体

1.       兔源磷酸STAT3 (Tyr705) (D3A7) (Cell Signalling 9145)

2.       小鼠Stat3 (124H6)抗体 (Cell Signalling 9139)

3.       HRP 羊抗兔  (BD Bioscicence 554021)

4.       兔源β-Actin (13E5)抗体  (Cell Signalling 4970)

5.       HRP偶联的小鼠IgG抗体  (Cell Signalling 7076)

备注:以上抗体均已检测,可以取代用户的抗体

II. 缓冲液及试剂

1.       1X 磷酸缓冲液(PBS).

2.       蛋白裂解缓冲液:M-PER哺乳类蛋白提取试剂 (Thermo Scientific 78501)1x添加了蛋白酶和磷酸酶抑制剂的蛋白酶抑制剂混合溶液

3.       Bio-Rad蛋白定量溶液

4.       电泳缓冲液:配制1L缓冲液:100ml 10x Tris/Glycine加入900ml 双蒸水

5.       转膜缓冲液:配制1L缓冲液:100ml 10x Tris/Glycine/SDS (BioRad 161-0772)200ml 甲醇 (Fisherscientific A412-20)加水700ml定容至1

6.       1X TBS :配制1L: 100ml 10x TBS 加入900ml 双蒸水

7.       封闭液: 1X TBS, 0.1% 吐温-20 (Santa cruz biotechnology, sc-29113) 加入 5% w/v BSA (MP Biomedicals 810033).

8.       洗膜缓冲: 1X TBS, 0.1% 吐温-20 (TBS/T).

9.       抗稀释缓冲液1X TBS, 0.1% 吐温-20 加入 5% BSA.

10.   持续增强超级信号底物 (Thermo Scientific 34075).

11.   高精度彩染蛋白分子量指示标准  (BioRad 161-0374)

12.   蛋白印迹剥膜缓冲液 (Thermo Scientific 46430).

13.   印迹用膜:本实验推荐使用硝酸纤维素膜,或者PVDF膜。

 

实验仪器

 

1.       Eppendorf 5415 R离心机 (Eppendorf)

2.       Thermolyne Rotomix 50800 (BioSurplus)

3.       SmartSpec Plus分光光度计 (BioRad)

 

实验步骤

 

1.       蛋白印迹

1)      用含有调节因子的新鲜培养基将细胞处理一定的时间。

2)      将培养基吸出,用1X PBS漂洗细胞,抽气

3)      加入1X 蛋白裂解缓冲液裂解细胞(见材料II-2)。迅速将细胞挂下并转移到离心管中,室温放置5分钟

4)      13000rpm离心10分钟.

5)      将上清转移到新的离心管中。

6)      Bio-Rad蛋白定量试剂检测蛋白浓度

7)      20 μg样品,95100°C加热10分钟,在冰上冷却

8)      20 μg样品和7 μl高精度彩染蛋白分子量指示标准SDS-PAGE(10 cm x 10 cm)上样以检测蛋白分子量。

9)      80V电压电泳直到蛋白指示剂移动到胶的底部。

10)  30V电压转膜,4°C 过夜

2.       膜的封闭以及STAT3磷酸化状态的检测

1)      将膜在25 ml 封闭液中孵育1小时。

2)      洗膜3,每次5分钟,用15ml TBS

3)      加入10ml封闭液稀释的抗(Phospho-Stat3 (Tyr705) (D3A7) XP? Rabbit antibody (1:1000))孵育膜,轻度震荡,4°C过夜

4)      洗膜3,每次5分钟,用15ml TBS

5)      加入10ml封闭液稀释的二抗(HRP-conjugated Goat Anti-Rabbit secondary antibody (1:5000))以及生物素标记的HRP偶联生物素抗体孵育膜,轻度震荡,室温1小时

6)      洗膜3,每次5分钟,用15ml TBS.

7)      加入4ml持续增强超级信号底物孵育膜,轻度震荡,室温5分钟 

8)      将膜上多余的溶液吸干,并用塑料膜将膜包裹起来,X-光显影,最初的曝光时间大约为10

3.       STAT3的检测

1)      洗膜3,每次5分钟,用15ml TBS.

2)      加入10ml 剥膜缓冲液孵育15分钟去除结合的抗体

3)      洗膜3,每次5分钟,用15ml TBS

4)      重复步骤11-18 ,以Stat3 (124H6) Mouse mAb 为一抗, HRP-偶联小鼠IgG 为二抗

4.       β-Actin的检测

1)      重复步骤19-22 检测β-Actin

5.       STAT3 活性的分析

1)      扫描X-光片并计算pSTAT3 STAT3条带的密度。通过以下公式来计算STAT3的活性:

2)      STAT3 活性的百分比 = (pSTAT3的密度)/( STAT3的密度+pSTAT3的密度) x100%

 

References:

 

1.        Fujimoto M., Naka T., Nakagawa R., Kawazoe Y., Morita Y., Tateishi A., Okumura K., Narazaki M., 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. Journal of Immunology 165(4): 1799-806. 

2.        Campbell C.L., Jiang Z., Savarese D.M., Savarese T.M. (2001). Increased expression of the interleukin-11 receptor and evidence of STAT3 activation in prostate carcinoma. American Journal of Pathology 158(1): 25-32. 

 

 

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How to cite this protocol: Zhang, H. (2011). Western Blot for Detecting Phosphorylated STAT3. Bio-protocol Bio101: e125. DOI: 10.21769/BioProtoc.125; Full Text



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8/31/2011 4:08:20 PM  

Anonymous Lin
Test institute

实验步骤--蛋白质印迹第10步 "30V电压转膜,4°C 过夜" 其中 过夜是个什么概念,大概多少小时?
实验步骤--蛋白质印迹第3步中 “迅速将细胞刮下并转移到离心管中” ,将细胞刮下来是否会弄伤细胞?由于有好几个点,对时间间隔有无特殊要求?他现在是贴壁的细胞,采用胰酶将细胞转移下来是否也可以?

8/31/2011 4:09:04 PM  

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。

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