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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, 21071 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] 这个实验方案将描述酵母蛋白激酶Sch9体外激酶活性检验的实验程序。这个规程可以适用于检测其它酵母蛋白激酶激酶活性。

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.

材料与试剂

 

1.         Tris  (C4H11NO3  Fisher 公司,货号 #77-86-1)

2.         氯化钠 (Fisher 科学,货号 # 7647-14-5)

3.         EDTA (Na2EDTA·2H2O, Sigma公司,货号 # ED2SS)

4.         曲通 X-100 (Fisher 科学,货号 # 9002-93-1)

5.         PMSF (苯甲基酰氟化物, C7H7FO2S, Sigma 公司,货号 # P7626)

6.         完全蛋白酶抑制剂混合物 (Roche 公司,货号 # 04693159001)

7.         PhosSTOP片剂 (Roche 公司,货号 # 04906837001)

8.         甘油 (Fisher 科学,货号 # 56-81-5)

9.         MgCl2  (USB 公司,货号 # 18641 500 GM)

10.     DTT (二硫苏糖醇) (Pierce 公司,货号# 20290)YPD

11.     雷帕霉素 (Santa Cruz公司,货号# sc-3504)

12.     玻璃珠 (Sigma公司,货号#G-8772)

13.     HA抗体 (12CA5) (Abcam公司,货号#

14.     ATP (Sigma公司,货号#.A2383-1G)

15.      [γ-32P]-ATP (PerkinElmer 公司,货号#BLU002250UC)

16.     考马斯蓝 R250 (National Diagnostics公司,货号# HS-605)

 

设备

 

1.         离心机

2.         混合器

3.         1.5 ml eppendorf 

4.         放射自显影设备

 

程序

 

1.         接种W303a野生型细胞或是含有带着它的激酶无活性形式(K441A) 和超活性形式(2D3E))pRS315-SCH9-HA3 载体的W303a 细胞, 30(300 rpm)10mL合成完全-组氨酸培养基中震荡摇晃培养过夜 

2.         2个含有100mL YPD 的瓶中分别次培养初始OD600=0.2酵母细胞,  30 °C剧烈摇荡培养直到酵母细胞的 OD600达到0.5(重要的是使用 YPD 而不是 SD 培养基).

3.         管中加入200 nM 雷帕霉素,另一管是作为空白对照。30 °C 剧烈摇荡培养 30 分钟。

4.         室温下 3000g减慢旋转收集细胞(重点是避免冷冻).

5.         弃大部分上清, 悬浮酵母细胞于残余培养基中并且分装于51.5 ml eppendorf 管中 (每管20 mL细胞培养物, 管中细胞过多会导致裂解不充分)

6.         收集细胞沉淀,立即加入200 uL冷冻的IP缓冲液和等量的玻璃珠。在4°C下立即用珠子敲打器裂解细胞1分钟(不要超过3分钟,否则激酶活性将由于过热而降低。在这之间敲打5次,每次15秒连同45秒敲打之间也会减弱 Sch9激酶活性)

7.         4℃下于20,000g减慢旋转10 min 收集细胞裂解物,混合裂解物。

8.         立即加入 1 μg HA 抗体 (12CA5) 1mg/500 μl 细胞裂解物,  4°C下轻柔的转动1 小时。 使用IP缓冲液冲洗蛋白A/G 3次,加入50 μl (100 μl 50% 研磨液)到(8)步骤中的细胞裂解物中,并且再次转动1小时。

9.         减慢旋转收集珠子,使用0.5 mL 冷冻 IP缓冲液冲洗3次。

10.     加入0.5 mL 1X 冷冻激酶缓冲液, 节余50 μl 置于另一个1.5 eppendorf 管中用于HA抗体的western blot 实验。

11.     减慢旋转并且在50 μL 激酶免疫混合物中加入100 μM ATP, 0.5 μg 细菌表达的GST-Maf1

12.     反应体系加入50μCi [γ-32P]-ATP,漩涡并且30°C 孵育15-30 分钟。

13.     加入2.5X SDS 上样染料100 °C 加热 5 min激酶反应活性被终止。

14.     半数样品用于 SDS-PAGE,使用考马斯蓝染色检测底物。

15.     凝胶干燥器80干燥胶2 小时。通过放色自显影显示磷酸化底物磷酸化。

 

配置试剂

 

1.         IP 缓冲液: 50mM Tris-盐酸 pH7.5, 150mM氯化钠 0.5 mM EDTA, 0.5 % 曲通X-100. 使用前加入2mM PMSF,罗氏完全蛋白酶抑制剂混合物phosSTOP 片剂。不同的氯化钠浓度或是 曲通 X-100 水平可获取不同激酶和不同抗体的最佳条件

2.         激酶缓冲液: 1X PBS (pH7.4)20% 甘油, 4mM MgCl2, 10mM DTT,蛋白酶抑制剂)

 

参考文献

 

1.         Wei Y., Zheng X.F. (2009). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-90. 

2.         Wei Y., Tsang C.K., Zheng X.F. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO Journal 28(15): 2220-30. 

 

 

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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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