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GTP Binding Assays in Arabidopsis thaliana
拟南芥的GTP结合试验   

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Abstract

Signaling through G proteins constitutes an ancient mechanism that functions in the transduction of extracellular signals into intracellular responses. Activation of a proper receptor by a stimuli leads to an exchange of GDP for GTP, the activation of G proteins and the dissociation of Gα-GTP from the Gβγ dimer for the heterotrimeric G proteins. The G protein subunits remain active until the intrinsic GTPase activity or/and a GTPase activating protein result in the hydrolysis of GTP to GDP and the inactivation of the protein. Here we describe a protocol for measuring GTP binding activity from Arabidopsis plant protein extracts using GTP γ35S. GTP γ35S assay measures the level of G protein activation following a stimuli, by determining the binding activity of the non-hydrolysable analog GTP γ35S. To determine specific G protein activities specific mutants and/or overexpressors extracts should be included and measured as controls.

Keywords: GTP binding(GTP结合), Arabidopsis(拟南芥), GPA1(属)

Materials and Reagents

  1. GTP γ35S 1,200 Ci/mmol (PerkinElmer, catalogue number: NEG030H250UC )
  2. 0.45 μm nitrocellulose filters (Bio-Rad, catalogue number: 162-0115 )
  3. Scintillation solution (Optiphase Hisafe 3, Perkin Elmer Inc, catalogue number: 1200-437 )
  4. Bradford reagent
  5. Triton X-100
  6. Tris HCl (pH 8)
  7. NaCl
  8. EDTA
  9. DTT (USB Corporation, Cleaveland OH USA, cataslogue number: 15397 )
  10. Roche complete protease inhibitor (Roche, Molecular Biochemicals)
  11. Extraction buffer (see Recipes)
  12. Reaction buffer (see Recipes)
  13. Initiation buffer (see Recipes)
  14. Stop buffer (see Recipes)

Equipment

  1. Liquid Scintillation Counter Wallac 1214 RackBeta (Pharmacia, Turku, Finland)

Procedure

I.  Protein extraction protocol (Keep all steps at 4 °C)

  1. Harvest approximately 300 mg of Arabidopsis tissue in liquid nitrogen.
  2. Homogenize with mortar and pestle to a fine powder.
  3. Resuspend the powder in 500 μl of extraction buffer (you can use manual homogeneizer).
  4. Centrifuge 15 min at 10,000 rpm at 4 °C and discard the pellet.
  5. Measure protein concentration in the supernatant as described (Bradford, 1976).

II.  GTP binding assay (reaction: final volume 200 μl)

  1. Mix 100 μg of proteins in 100 μl extraction buffer with 80 μl of reaction buffer.
  2. Add 20 μl of Initiation Buffer.
  3. Expose to the corresponding stimuli (Light, agonist etc).
    (You should adjust the specific stimuli, time of exposition, concentration, intensity etc where you can detect optimum binding).
  4. Incubate the reaction 10 min at room temperature.
  5. Add 2 ml of ice-cold stop buffer to finish the reaction.
  6. Filter the reaction through 0.45 μm nitrocellulose filters using vacuum. We use 3 cm diameter disks.
  7. Wash the disk filters 5 times with 2 ml of cold Stop Buffer using vacuum. This step is important to wash away unbound GTP γ35S.
  8. Dry disk filters containing the membranes for 15 min at 75 °C.
  9. Put the disks in plastic vials containing 2 ml of scintillation solution.
  10. Record the cpm in the 35S energy range with a liquid scintillation counter.
  11. Important: Unspecific binding must be estimated using blank reactions. We recommend three replicates of two possible blanks: reactions without proteins (reaction buffer plus initiation buffer) or 100 μg of protein samples preheated to 95 °C for 10 min. We did not find differences in GTP binding between both blanks.
  12. Subtract the average cpm obtained with the blank samples from the cpm obtained for each measured sample to obtain the correct value.

Recipes

  1. Extraction Buffer
    50 mM Tris HCl pH 8
    100 mM NaCl
    1 mM EDTA
    1 mM DTT
    0.1% Triton X-100
    1x Roche complete protease inhibitor
  2. Reaction buffer (use 80 μl from the stock per reaction)
    Stock
    Final concentration in 200 μl reaction
    50 mM Tris HCl (pH 8)
    20 mM Tris HCl (pH 8)
    250 mM NaCl
    100 mM NaCl
    2.5 mM EDTA
    1 mM EDTA
    2.5 mM DTT
    1 mM DTT
    0.25% Triton X-100
    0.1% Triton X-100
  3. Initiation buffer (use 20 μl from the Stock per reaction) (pH 8)
    Stock
    Final concentration in 200 μl reaction
    100 mM MgCl2
    10 mM MgCl2
    1% Triton X-100
    0.1% Triton X-100
    Add GTP γ35S, 300,000 cpm/reaction.
  4. Stop buffer (use 2 ml per reaction)
    20 mM Tris-HCl (pH 8)
    100 mM NaCl
    25 mM MgCl2
    1 mM phosphate buffer

Acknowledgments

This protocol was adapted from Fox et al. (2012). This work was financially supported by FonCyT PICT 2010 num 1821 to A.M.

References

  1. Fox, A. R., Soto, G. C., Jones, A. M., Casal, J. J., Muschietti, J. P. and Mazzella, M. A. (2012). cry1 and GPA1 signaling genetically interact in hook opening and anthocyanin synthesis in Arabidopsis. Plant Mol Biol 80(3): 315-324.

简介

通过G蛋白的信号传导构成了在将细胞外信号转导到细胞内反应中起作用的古老机制。通过刺激激活适当的受体导致GTP的GDP交换,G蛋白的激活和G emα-GTP从G emβγ二聚体的解离异源三聚体G蛋白。 G蛋白亚基保持活性,直到内在的GTP酶活性或/和GTP酶活化蛋白导致GTP水解为GDP和蛋白质的失活。在这里,我们描述了使用GTPγ S,从拟南芥植物蛋白提取物测量GTP结合活性的方案。 GTPγ测定通过测定不可水解的类似物GTPγ35 S的结合活性来测量刺激后G蛋白活化的水平。为了确定特异性G蛋白活性,应包括特异性突变体和/或过表达体提取物,并作为对照测量。

关键字:GTP结合, 拟南芥, 属

材料和试剂

  1. GTP 35 (PerkinElmer,目录号:NEG030H250UC)
  2. 0.45μm硝化纤维素过滤器(Bio-Rad,目录号:162-0115)
  3. 闪烁溶液(Optiphase Hisafe 3,Perkin Elmer Inc,目录号:1200-437)
  4. Bradford试剂
  5. Triton X-100
  6. Tris HCl(pH 8)
  7. NaCl
  8. EDTA
  9. DTT(USB Corporation,Cleaveland OH USA,cataslogue number:15397)
  10. 罗氏完全蛋白酶抑制剂(Roche,Molecular Biochemicals)
  11. 提取缓冲区(参见配方)
  12. 反应缓冲液(参见配方)
  13. 启动缓冲区(请参阅配方)
  14. 停止缓冲液(参见配方)

设备

  1. 液体闪烁计数器Wallac 1214 RackBeta(Pharmacia,Turku,Finland)

程序

I.  蛋白质提取方案(保持所有步骤在4°C)

  1. 在液氮中收获约300mg拟南芥组织
  2. 用研钵和杵将其均匀化成细粉
  3. 将粉末重悬于500μl提取缓冲液中(您可以使用手动均质器)
  4. 在4℃下以10,000rpm离心15分钟,弃去沉淀
  5. 如上所述测量上清液中的蛋白质浓度(Bradford,1976)

II。  GTP结合测定(反应:终体积200μl)

  1. 混合100微升蛋白质在100微升提取缓冲液与80微升反应缓冲液
  2. 加入20μl起始缓冲液。
  3. 暴露于相应的刺激(光,激动剂等)。
    (你应该调整特定的刺激,暴露时间,浓度,强度等,在哪里可以检测到最佳绑定)
  4. 在室温下孵育反应10分钟。
  5. 加入2ml冰冷的终止缓冲液以完成反应
  6. 使用真空通过0.45μm硝化纤维素过滤器过滤反应。 我们使用3厘米直径的磁盘
  7. 使用真空用2ml冷停止缓冲液洗涤磁盘过滤器5次。 该步骤对于洗去未结合的GTPγ 35 S是重要的
  8. 干膜过滤器,在75℃下包含膜15分钟
  9. 将磁盘放在含有2毫升闪烁溶液的塑料小瓶中。
  10. 使用液体闪烁计数器记录 S能量范围中的cpm。
  11. 重要:非特异性结合必须使用空白反应估计。 我们建议两个可能空白的三个重复:没有蛋白质的反应(反应缓冲液加启动缓冲液)或100μg预热至95℃10分钟的蛋白质样品。 我们在两个空白之间没有发现GTP结合的差异。
  12. 从每个测量样品获得的cpm中减去空白样品获得的平均cpm,得到正确的值。

食谱

  1. 提取缓冲区
    50mM Tris HCl pH 8
    100 mM NaCl
    1mM EDTA
    1 mM DTT
    0.1%Triton X-100 1×Roche完全蛋白酶抑制剂
  2. 反应缓冲液(每次反应使用80μl来自原料)
    库存
    最终浓度为200μl反应物
    50mM Tris HCl(pH8)
    20mM Tris HCl(pH8)
    250mM NaCl 100 mM NaCl
    2.5mM EDTA
    1mM EDTA
    2.5mM DTT
    1 mM DTT
    0.25%Triton X-100 0.1%Triton X-100
  3. 起始缓冲液(使用来自每次反应的20μl)(pH 8)
    库存
    最终浓度为200μl反应物
    100mM MgCl 2/v/v 10mM MgCl 2/
    1%Triton X-100 0.1%Triton X-100
    加入GTPγ S,300,000 cpm /反应
  4. 停止缓冲液(每次反应使用2ml)
    20mM Tris-HCl(pH8)
    100 mM NaCl
    25mM MgCl 2·h/v 1mM磷酸盐缓冲液

致谢

该方案改编自Fox等人(2012)。 这项工作得到了FonCyT PICT 2010年第1821号到A.M.

参考文献

  1. Fox,A.R.,Soto,G.C.,Jones,A.M.,Casal,J.J.,Muschietti, 和Mazzella,M.A。(2012)。 cry1和GPA1信号遗传相互作用   在拟南芥中的钩开启和花青素合成 。 植物分子 Biol.80(3):315-324。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fox, A. R. and Mazzella, M. A. (2013). GTP Binding Assays in Arabidopsis thaliana. Bio-protocol 3(9): e752. DOI: 10.21769/BioProtoc.752.
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