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This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase.

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In vitro Protein Kinase Assay
蛋白激酶的体外实验

微生物学 > 微生物生物化学 > 蛋白质 > 活性
作者: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
Vol 2, Iss 11, 6/5/2012, 23072 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase.

[Abstract] 该方案将描述酵母蛋白激酶Sch9的体外激酶测定的实验程序。 此协议可以定制,以检测其他酵母蛋白激酶的激酶活性。

Materials and Reagents

  1. W303a wild type yeast cells
  2. Tris base (C4H11NO3) (Thermo Fisher Scientific, catalog number: 77-86-1 )
  3. NaCl (Thermo Fisher Scientific, catalog number: 7647-14-5 )
  4. Na2EDTA•2H2O (EDTA) (Sigma-Aldrich, catalog number: ED2SS )
  5. Triton X-100 (Thermo Fisher Scientific, catalog number: 9002-93-1 )
  6. Phenylmethanesulfonyl fluoride (PMSF) (C7H7FO2S) (Sigma-Aldrich, catalog number: P7626 )
  7. Complete protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693159001 )
  8. PhosSTOP tablet (F. Hoffmann-La Roche, catalog number: 04906837001 )
  9. Glycerol (Thermo Fisher Scientific, catalog number: 56-81-5 )
  10. MgCl2 (USB, catalog number: 18641 500 GM )
  11. Dithiothreitol (DTT) (Thermo Fisher Scientific, Pierce Antibodies, catalog number: 20290 )
  12. Rapamycin (Santa Cruz Biotechnology, catalog number: sc-3504 )
  13. Glass beads (Sigma-Aldrich, catalog number: G-8772 )
  14. HA antibody (12CA5) (Abcam, catalog number: ab16918
  15. ATP (Sigma-Aldrich, catalog number: A2383-1G )
  16.  [γ-32P]-ATP (PerkinElmer, catalog number: BLU002250UC )
  17. Coomassie Blue R250 (National Diagnostics, catalog number: HS-605 )
  18. HCl
  19. 2.5x SDS loading dye
  20. IP buffer (see Recipes)
  21. Kinase buffer (see Recipes)
  22. PBS buffer (see Recipes)

Equipment

  1. Standard bench-top centrifuge
  2. Shaker
  3. 1.5 ml Eppendorf tubes
  4. Authoradiograph

Procedure

  1. Inoculate W303a wild type or W303a cells containing vector, pRS315-SCH9-HA3 and its kinase dead form (K441A) and hyperactive form (2D3E) overnight in 10 ml SC-His- medium. Shaking vigorously (300 rpm) at 30 °C.
  2. Subculture yeast cells in 2 flasks containing 100 ml YPD each with starting OD600=0.2.
  3. Shake vigorously at 30 °C to OD600=0.5 (important to use YPD rather than SD medium).
  4. Add to one flask 200 nM of rapamycin and another vehicle control. Shake vigorously at 30 °C for 30 min.
  5. Spin down at room temperature (RT) at 3,000 x g to collect cells (It is important to avoid freezing).
  6. Discard most of the supernatant, then suspend yeast cells in the remaining medium and split into 5x 1.5 ml Eppendorf tubes (20 ml cell culture/tube, more cells in one tube do not breakdown sufficiently).
  7. Collect cell pellets, immediately add 200 µl ice-cold IP buffer and the same amount of glass beads.
  8. Immediately breakdown cells by beads beater at 4 °C for 1 min. Note: Do not exceed 3 min, otherwise kinase activity will begin to decrease due to overheating. 15 sec x 5 beating with 45 sec in between also decreases Sch9 kinase activity.
  9. Collect cell lysate by spinning down at 20,000 x g, 10 min at 4 °C. Combine lysate.
  10. Immediately add 1 µg HA antibody (12CA5) to 1 mg/500 μl cell lysate, gently rotate at 4 °C for 1 h.
  11. Wash protein A/G beads 3x with IP buffer. Add 50 μl (100 μl 50% slurry) to cell lysate and further rotate for another 1 h.
  12. Spin down and collect beads. Wash 3x with 0.5 ml ice-cold IP buffer.
  13. Add 0.5 ml 1x with ice-cold kinase buffer. Save 50 µl in another 1.5 ml Eppendorf tube for western blot with HA antibody.
  14. Spin down and add to the immunocomplex 100 µM ATP, 0.5 µg bacterially-expressed GST-Maf1 in 50 µl kinase.
  15. Add to the reaction system 50 µCi [γ-32P]-ATP. Vortex and incubate at 30 °C for 15-30 min.
  16. Kinase reaction was stopped by heating at 100 °C for 5 min in 2.5x SDS loading dye.
  17. Half of the sample should be subjected to SDS-PAGE. Substrate will be detected by Coomassie blue staining.
  18. Dry the gel using gel dryer at 80 °C for 2 h. Phosphorylation of substrate is revealed by authoradiograph.

Recipes

  1. IP buffer
    50 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    0.5 mM EDTA
    0.5% Triton X-100
    Add 2 mM PMSF, Roche Complete protease inhibitor cocktail and phosSTOP tablet before use. Vary NaCl concentration or/and Triton X-100 level to obtain optimum condition for different kinase and different antibodies.
  2. 1x PBS (pH 7.4)
  3. Kinase buffer
    1x PBS (pH 7.4)
    20% glycerol
    4 mM MgCl2
    10 mM DTT
    Protease inhibitor

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.

材料和试剂

  1. W303a野生型酵母细胞
  2. Tris碱(C 4 H 11 NO 3)(Thermo Fisher Scientific,目录号:77-86-1)
  3. NaCl(Thermo Fisher Scientific,目录号:7647-14-5)
  4. Na 2 EDTA·2H 2 O(EDTA)(Sigma-Aldrich,目录号:ED2SS)
  5. Triton X-100(Thermo Fisher Scientific,目录号:9002-93-1)
  6. 苯基甲磺酰氟(PMSF)(C 7 H 7 SO 4 S)(Sigma-Aldrich,目录号:P7626)
  7. 完全蛋白酶抑制剂混合物(F.Hoffmann-La Roche,目录号:04693159001)
  8. PhosSTOP片剂(F.Hoffmann-La Roche,目录号:04906837001)
  9. 甘油(Thermo Fisher Scientific,目录号:56-81-5)
  10. MgCl 2(USB,目录号:18641 500 GM)
  11. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,Pierce Antibodies,目录号:20290)
  12. 雷帕霉素(Santa Cruz Biotechnology,目录号:sc-3504)
  13. 玻璃珠(Sigma-Aldrich,目录号:G-8772)
  14. HA抗体(12CA5)(Abcam,目录号:ab16918
  15. ATP(Sigma-Aldrich,目录号:A2383-1G)
  16.   [γ- 32 P] -ATP(PerkinElmer,目录号:BLU002250UC)
  17. 考马斯蓝R250(National Diagnostics,目录号:HS-605)
  18. HCl
  19. 2.5x SDS装填染料
  20. IP缓冲区(参见配方)
  21. 激酶缓冲液(参见配方)
  22. PBS缓冲液(见配方)

设备

  1. 标准台式离心机
  2. 振动器
  3. 1.5 ml Eppendorf管
  4. 作者日历

程序

  1. 接种含有载体 pRS315-SCH9-HA3 及其激酶死亡形式(K441A)和多动型(2D3E)的W303a野生型或W303a细胞 在10ml SC-His-培养基中过夜。 在30℃下剧烈振荡(300rpm)。
  2. 在含有100ml YPD的2个烧瓶中传代酵母细胞,起始OD 600 = 0.2。
  3. 在30℃下剧烈振摇至OD 600 = 0.5(使用YPD而不是SD培养基是重要的)。
  4. 向一个烧瓶中加入200nM雷帕霉素和另一种媒介物对照。 在30℃下剧烈振荡30分钟
  5. 在室温(RT)下以3,000xg 旋转以收集细胞(重要的是避免冻结)。
  6. 弃去大部分上清液,然后将酵母细胞悬浮在剩余的培养基中,并分成5×1.5ml Eppendorf管(20ml细胞培养物/管,一个管中的更多细胞未充分分解)。
  7. 收集细胞沉淀,立即加入200μl冰冷的IP缓冲液和相同量的玻璃珠。
  8. 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,否则激酶活性会由于过热而开始下降。 15秒×5跳动与45秒之间也降低Sch9激酶活性。
  9. 通过在20000×g下旋转收集细胞裂解物,在4℃下10分钟。 组合裂解物。
  10. 立即向1mg /500μl细胞裂解液中加入1μgHA抗体(12CA5),在4℃下轻轻旋转1小时。
  11. 用IP缓冲液洗涤蛋白A/G珠3x。 加入50微升(100微升50%浆液)细胞裂解液,进一步旋转1小时
  12. 旋转和收集珠。 用0.5ml冰冷的IP缓冲液洗涤3次。
  13. 加入0.5 ml 1x冰冷的激酶缓冲液。 保存50微升在另一个1.5毫升的Eppendorf管用于与HA抗体的western印迹
  14. 离心并加入免疫复合物100μMATP,0.5μg细菌表达的GST-Maf1在50μl激酶。
  15. 向反应体系中加入50μCi[γ-32 P] -ATP。 涡旋并在30℃孵育15-30分钟
  16. 通过在2.5x SDS加载染料中在100℃加热5分钟来终止激酶反应
  17. 一半的样品应进行SDS-PAGE。 底物将通过考马斯蓝染色检测
  18. 使用凝胶干燥器在80℃下干燥凝胶2小时。 基质的磷酸化通过作者自显示

食谱

  1. IP缓冲区
    50mM Tris-HCl(pH7.5) 150mM NaCl 0.5mM EDTA 0.5%Triton X-100 在使用前加入2mM PMSF,Roche完全蛋白酶抑制剂混合物和phosSTOP片剂。 改变NaCl浓度或/和Triton X-100水平以获得不同激酶和不同抗体的最佳条件。
  2. 1x PBS(pH 7.4)
  3. 激酶缓冲液
    1x PBS(pH 7.4)
    20%甘油 4mM MgCl 2/
    10 mM DTT
    蛋白酶抑制剂

致谢

该方案改编自并用于Wei和Zheng(2009)和Wei et al。 (2009)。

参考文献

  1. Wei,Y。和Zheng,X.F。(2009)。 Sch9部分介导TORC1信号转导以控制核糖体RNA合成。 细胞周期 8(24):4085-4090。
  2. Wei,Y.,Tsang,C.K.and Zheng,X.F。(2009)。 TORC1调控RNA聚合酶III依赖性转录的机制。 EMBO J 28(15):2220-2230
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How to cite this protocol: Wei, Y. (2012). In vitro Protein Kinase Assay. Bio-protocol 2(11): e212. DOI: 10.21769/BioProtoc.212; Full Text



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