搜索

An Improved Method for PAGE-based Detection of Phosphorylated Protein in Yeast
一种基于PAGE的检测酵母磷酸化蛋白的改进技术   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

One dimensional polyacrylamide gel electrophoresis has been successfully used to detect protein phosphorylation. This method is very simple and highly reproducible. Hyperhosphorylated proteins usually migrate slowlier than dephosphorylated proteins. However, not all phosphorylated proteins can be readily detected, due to sub-optimal sample preparation and electrophoresis conditions. Here, an improved method is described that can detect phosphorylation of yeast proteins ranging from 15 kD to 200 kD. The improvement in gel electrophoresis should also be applicable to mammalian culture cells.

Keywords: Phosphorylation (磷酸化), Yeast (酵母), Mobility shift (迁移), Acrylamide gel (丙烯酰胺凝胶), Western blot (免疫印迹 (参照中文category))

Materials and Reagents

  1. Tris base (C4H11NO3) (Thermo Fisher Scientific, catalog number: 77-86-1 )
  2. NaCl (Thermo Fisher Scientific, catalog number: 7647-14-5 )
  3. EDTA (Na2EDTA•2H2O) (Sigma-Aldrich, catalog number: ED2SS )
  4. Triton X-100 (Thermo Fisher Scientific, catalog number: 9002-93-1 )
  5. Phenylmethanesulfonyl fluoride (PMSF) (C7H7FO2S) (Sigma-Aldrich, catalog number: P7626 )
  6. Complete protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693159001 )
  7. PhosSTOP tablet (F. Hoffmann-La Roche, catalog number: 04906837001 )
  8. SDS [CH3 (CH2)11OSO3Na] (Sigma-Aldrich, catalog number: L3771 )
  9. Glycerol (Thermo Fisher Scientific, catalog number: 56-81-5 )
  10. β-mercaptoethanol (Sigma-Aldrich, catalog number: M7154 )
  11. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  12. Synthetic complete (SC) medium
  13. Rapamycin
  14. Methanol
  15. 5x SDS loading buffer (see Recipes)
  16. Yeast cell lysis buffer (see Recipes)

Equipment

  1. Standard bench-top centrifuge
  2. Sterilized toothpick
  3. Incubator
  4. Shaker
  5. 1.5 ml eppendorf tubes
  6. 25G needle
  7. Western blot equipment
  8. Refrigerator

Procedure

  1. Streak out yeast cells from -80 °C freezer stocks by using a sterilized toothpick, incubate at 30 °C incubator for around 2 days in standard yeast growth media plates.
  2. Inoculate single colony into YPD or synthetic complete (SC) medium overnight at 30 °C shaker.
  3. Subculture yeast cells from OD600=0.1, continue to incubate at 30 °C shaker for around 4-6 h until OD600 =0.4.
  4. To treat with rapamycin, final concentration of 200 nM rapamycin or drug vehicle (methanol) was added to cell cultures for 1 h or as otherwise indicated.
  5. Collect yeast cells by spinning down at 3,000 x g for 15 sec without freezing on ice. 
    Note: As long as yeast cells remain in YPD and room temperature, phosphorylation should be expected to be the same. Freezing on ice for 10 min tends to mitigate phosphorylation in my hands. Washing with water or PBS buffer also decreases phosphorylation.
  6. Discard most of the supernatant.
  7. Suspend yeast cells in the remaining medium and split into 1.5 ml eppendorf tubes.
  8. Spin down at room temperature at 3,000 x g 15 sec.
  9. Collect cell pellets, immediately add 100 μl (for 10 ml cells OD600=0.4) ice-cold cells lysis buffer and the same amount of ice-cold beads. Immediately breakdown cells by a beads beater at 4°C for 1 min.
    Note: Do not exceed 3 min, otherwise protein will begin to dephosphorylate due to overheating. 15 sec x5 beating with 45 sec in between also decreases phosphorylation.
  10. If used for western blot directly, add 25 μl 5x SDS loading buffer and vortex for a few seconds.
  11. Pore the bottom of the tube with 25G needle (heating in flame) and place on the top of a new eppendorf tube.
  12. Spin down at 3,000 x g for 5 min (the mild spindown will retain some chromatin-associated or strong membrane-bound proteins, which could be phosphorylated form).
  13. For assays such as phosphatase treatment (CIP assay), lysate should be used immediately without freezing-thawing.
  14. For western blot, collect 80 μl lysate and add 20 μl 5x SDS loading dye, place on 100 °C heat blot for 5 min. Samples may be stored at -20 °C for 2 months, -80 °C for up to 6 months.
  15. Run suggested concentration of SDS polyacrylamide gel according to protein molecular weight. Try 50, 100, 150 and 200 volts to detect phosphorylated proteins by using a known phosphorylated protein as positive control.
    Note: Phosphorylation of Maf1 cannot be detected by varying acrylamide: bis-arylamide ratio, rather by increasing ruing voltage to 150 volts.

Recipes

  1. Yeast cell lysis buffer
    50 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    0.5 mM EDTA
    1% Triton X-100
    2mM PMSF
    Roche Complete protease inhibitor cocktail
    PhosSTOP tablet
    Put on ice before use.
  2. 5x SDS loading buffer
    60 mM Tris-HCl (pH 6.8)
    2% SDS
    10% glycerol
    5% β-mercaptoethanol
    0.01% bromophenol blue

Acknowledgments

This protocol was adapted from and used in Wei and Zheng (2009) and Wei et al. (2009).

References

  1. Wei, Y. and Zheng, X. F. (2009). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090.
  2. Wei, Y., Tsang, C. K. and Zheng, X. F. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28(15): 2220-2230.

简介

已经成功地使用一维聚丙烯酰胺凝胶电泳来检测蛋白质磷酸化。 这种方法非常简单和高度可重现。 高磷酸化蛋白通常比去磷酸化蛋白更慢地迁移。 然而,由于次优的样品制备和电泳条件,不是所有的磷酸化蛋白都可以容易地检测。 这里,描述了一种改进的方法,其可以检测范围从15kD至200kD的酵母蛋白质的磷酸化。 凝胶电泳的改进也应适用于哺乳动物培养细胞。

关键字:磷酸化, 酵母, 迁移, 丙烯酰胺凝胶, 免疫印迹 (参照中文category)

材料和试剂

  1. Tris碱(C 4 H 11 NO 3)(Thermo Fisher Scientific,目录号:77-86-1)
  2. NaCl(Thermo Fisher Scientific,目录号:7647-14-5)
  3. EDTA(Na 2 EDTA; 2H 2 O)(Sigma-Aldrich,目录号:ED2SS)
  4. Triton X-100(Thermo Fisher Scientific,目录号:9002-93-1)
  5. 苯基甲磺酰氟(PMSF)(C 7 H 7 SO 4 S)(Sigma-Aldrich,目录号:P7626)
  6. 完全蛋白酶抑制剂混合物(F.Hoffmann-La Roche,目录号:04693159001)
  7. PhosSTOP片剂(F.Hoffmann-La Roche,目录号:04906837001)
  8. SDS [CH 3(CH 2)11] OSO 3 [Na](Sigma-Aldrich,目录号:L3771 )
  9. 甘油(Thermo Fisher Scientific,目录号:56-81-5)
  10. β-巯基乙醇(Sigma-Aldrich,目录号:M7154)
  11. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  12. 合成完全(SC)培养基
  13. 雷帕霉素
  14. 甲醇
  15. 5x SDS加载缓冲液(参见配方)
  16. 酵母细胞裂解缓冲液(见配方)

设备

  1. 标准台式离心机
  2. 灭菌牙签
  3. 孵化器
  4. 振动器
  5. 1.5 ml eppendorf管
  6. 25G针
  7. Western印迹设备
  8. 冰箱

程序

  1. 使用无菌牙签将酵母细胞从-80℃冷冻库中分离,在标准酵母生长培养基平板中在30℃培养箱中孵育约2天。
  2. 在30℃振荡器中将单菌落接种到YPD或合成完全(SC)培养基中过夜
  3. 将来自OD 600 = 0.1的传代酵母细胞继续在30℃振荡器中孵育约4-6小时,直到OD 600 = 0.4。
  4. 为了用雷帕霉素处理,将最终浓度为200nM的雷帕霉素或药物载体(甲醇)加入细胞培养物中1小时或如其他指示的。
  5. 收集酵母细胞,通过在3000×g下旋转15秒钟,而不在冰冻结。
    注意:只要酵母细胞保持在YPD和室温下,磷酸化应该预期是相同的。在冰上冷冻10分钟倾向于减轻我手中的磷酸化。用水或PBS缓冲液洗涤也降低磷酸化。
  6. 弃去大部分上清液。
  7. 将酵母细胞悬浮在剩余的培养基中,并分成1.5ml eppendorf管
  8. 在室温下以3,000×g /秒旋转15秒。
  9. 收集细胞沉淀,立即加入100μl(对于10ml细胞OD 600 = 0.4)冰冷的细胞裂解缓冲液和相同量的冰冷的珠子。立即通过珠磨机在4℃下破碎细胞1分钟。
    注意:不要超过3分钟,否则蛋白质会由于过热而开始脱磷酸。 15秒×5次跳动,45秒钟之间也会降低磷酸化。
  10. 如果直接用于Western印迹,加入25μl5x SDS加样缓冲液,涡旋几秒钟
  11. 用25G针(在火焰中加热)对管的底部进行孔,并放置在新的埃彭道夫管的顶部。
  12. 在3,000×em下旋转5分钟(轻微的spindown将保留一些染色质相关的或强的膜结合蛋白,其可以是磷酸化形式)。
  13. 对于诸如磷酸酶处理(CIP测定)的测定,应当立即使用裂解物而不冻融
  14. 对于蛋白质印迹,收集80μl裂解物并加入20μl5x SDS加样染料,置于100℃加热印迹5分钟。 样品可以在-20℃保存2个月,-80℃保存6个月
  15. 根据蛋白质分子量运行建议浓度的SDS聚丙烯酰胺凝胶。 尝试50,100,150和200伏,通过使用已知的磷酸化蛋白作为阳性对照来检测磷酸化蛋白。
    注意:Maf1的磷酸化不能通过改变丙烯酰胺:双芳基酰胺的比率来检测,而是通过将升高的电压提高到150伏来检测。

食谱

  1. 酵母细胞裂解缓冲液
    50mM Tris-HCl(pH7.5) 150mM NaCl 0.5mM EDTA 1%Triton X-100 2mM PMSF
    Roche完全蛋白酶抑制剂混合物
    PhosSTOP平板电脑
    使用前请放在冰上。
  2. 5x SDS加样缓冲液
    60 mM Tris-HCl(pH 6.8)
    2%SDS
    10%甘油 5%β-巯基乙醇 0.01%溴酚蓝

致谢

该方案从Wei和Zheng(2009)和Wei等人(2009)改编并使用。

参考文献

  1. Wei,Y。和Zheng,X.F。(2009)。 Sch9 部分介导TORC1信号传导以控制核糖体RNA合成。 Cell Cycle 8(24):4085-4090。
  2. Wei,Y.,Tsang,C.K.and Zheng,X.F。(2009)。 通过TORC1调节RNA聚合酶III依赖性转录的机制 。 EMBO J 28(15):2220-2230。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wei, Y. (2012). An Improved Method for PAGE-based Detection of Phosphorylated Protein in Yeast. Bio-protocol 2(12): e210. DOI: 10.21769/BioProtoc.210.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。