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pERK Detection Assays Using the Surefire AlphaScreen® Kit (TGR Biosciences and PerkinElmer)
采用Surefire AlphaScreen® 试剂盒((TGR Biosciences and PerkinElmer)进行pERK检测   

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Cheng Zhang Cheng Zhang
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Abstract

Extracellular signal-regulated kinase 1 and 2 (ERK1/2) are serine/threonine protein kinases that are phosphorylated on Thr202/Tyr204 (ERK1) and Thr185/Tyr187 (ERK2). Phosphorylation of ERK1/2 (pERK1/2) arises from multiple stimuli, resulting in physiological responses that include cell growth, proliferation and differentiation. This protocol has been optimized for the detection of ligand-mediated pERK1/2 in adherent immortal cell lines expressing G protein-coupled receptors (GPCRs).

Materials and Reagents

  1. Dulbecco’s modified eagle medium (DMEM) (Life Technologies, Gibco®, catalog number: 11995-065 )
  2. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  3. SureFire® Reagents (Includes Lysis, Activation and Reaction buffers) (TGR BioSciences, catalog number: TGRES500 )
  4. AlphaScreen® General IgG (Protein A) detection kit (PerkinElmer, catalog number: 6760617 )
  5. White ProxiPlate 384-well microplate (PerkinElmer, catalog number: 6008280 )
  6. TopSeal (PerkinElmer, catalog number: 6005250 )
  7. NaCl
  8. KCl
  9. Na2HPO4
  10. KH2PO4
  11. Phosphate buffered saline (PBS) (see Recipes)
  12. Detection buffer (see Recipes)

Equipment

  1. Fusion-α plate reader or Envision plate reader with appropriate Alphascreen detection modules (PerkinElmer)
  2. Sterile 96-well clear flat bottom plates (BD Biosciences, Falcon®, catalog number: 353072 )
  3. Humidified incubator
  4. Multichannel pipettes
  5. Micropipettes
  6. Orbital shaker

Procedure

Notes:
1) Cells can either be stably or transiently expressing receptor of interest.
2) It is recommended to first perform a timecourse analysis to determine the time at which ligand-mediated pERK1/2 is maximal. For this, follow the same protocolusing a single concentration of ligand  (recommended concentration 100x Kd).
    Recommended initial timecourse (min): 90, 60, 45, 30, 15, 10, 8, 6, 4, 2, 1, 0.
3) Subsequent timecourses can then be refined to determine the precise time at which maximum ligand-induced pERK1/2 occurs.


I.   Cell preparation

Seed cells in suitable nutrient media (e.g. DMEM, 10% FBS, no antibiotics) into a sterile 96-well plate and incubate in a humidified environment at 37 °C, 5% CO2 to be ~90% confluent the following day (~24 h).

Note: Optimization for cell number depending on the cell line used will be necessary (we suggest a starting range of 10,000-50,000 cells per well). Recommended density for CHO FlpIN cells is 30,000 cells/ well.


II.  Stimulation

  1. The day following seeding, aspirate nutrient media, rinse once with 100 μl PBS, and replace with 90 μl prewarmed DMEM (no FBS).
  2. Incubate in a humidified environment at 37 °C, 5% CO2 for a minimum of 4 h (recommended 6 h, up to overnight (O/N)).
  3. Prepare serial dilutions of ligands at 10x final concentration in DMEM, enough for 10 μl/well, to be performed in duplicate (minimum), and enough for the number of timepoints if doing a timecourse.
    Note: Concentration range to use will depend on ligand affinity for receptor. For initial timecourse test, select a concentration 100x Kd of ligand and a DMEM control. The concentrations can then be refined in subsequent experiments. If using a peptide or ‘sticky’ ligand, prepare serial dilutions in DMEM with 0.1% BSA.
  4. Prepare a suitable concentration of FBS in DMEM, enough for 10 μl/well, to be performed in duplicate (minimum), and enough for the number of timepoints if doing a timecourse.
    Note: This is the internal control for the experiment – FBS promotes pERK1/2. Recommended final concentration of FBS in a CHO FlpIN cell line is 3-10%.
  5. Following preincubation in DMEM, add 10 μl of 10x prepared ligands or FBS to cells for a total volume of 100 μl, 1x final concentration.
    Note: For initial timecourse, begin at 90 min, and add ligand or FBS to cells at each timepoint until time 0 (no addition). For concentration response, add ligand or FBS at time of maximal induced pERK1/2 as determined through timecourse.
  6. After completion of stimulation, rapidly remove ligand-containing media from cells.
    Note: Depending on the cell type, this may involve flicking or gentle aspiration.
  7. Add 50 μl 1x Surefire® Lysis buffer.
    Note: Optimization for lysis volume will be necessary, and depends on the cell type, expression level of the receptor and efficiency of coupling to pERK1/2 pathways. Recommended starting lysis volume in a CHO FlpIN cell line is 30-100 μl.
  8. Incubate lysates at room temperature (RT) for 5-10 min on an orbital shaker.


III. Detection

  1. In reduced lighting conditions, prepare detection buffer.
  2. Transfer 5 μl of cell lysate from each well to a 384-well ProxiPlate.
  3. In reduced lighting conditions, add 8.5 μl Detection buffer to each sample.
  4. Seal the plate with TopSeal and wrap in foil.
    Note: Small volumes are subject to evaporation, TopSeal is essential.
  5. Incubate at RT for 2 h or 37 °C for 1 h in reduced lighting conditions.
    Note: If incubating at 37 °C, ensure the plate has returned to RT before measuring luminescence (~15 min at RT following 37 °C incubation should suffice). Detection beads are temperature sensitive.
  6. Analyse luminescence on a Fusion-α or Envision plate reader using standard α-screen settings.


IV. Data analysis

Data should be normalized to the response elicited by the FBS control.

Recipes

  1. Phosphate Buffered Saline (PBS)
    137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4.
  2. Detection buffer
    85.0% SureFire® reaction buffer
    14.2% SureFire® activation buffer*
    0.4% Acceptor beads
    0.4% Donor beads
    * Activation buffer should be stored at 4 °C, however, precipitation will occur at this temperature. Before use, heat to 37 °C to ensure all is dissolved.
    Prepare Detection buffer immediately before use. Discard unused detection buffer. Mix detection buffer gently. Do not vortex.
    Additional note: Lysates may be stored at -20 °C and pERK1/2 detected at a later time, but no longer than 2 weeks following stimulation.

References

  1. Koole, C., Wootten, D., Simms, J., Valant, C., Sridhar, R., Woodman, O. L., Miller, L. J., Summers, R. J., Christopoulos, A. and Sexton, P. M. (2010). Allosteric ligands of the glucagon-like peptide 1 receptor (GLP-1R) differentially modulate endogenous and exogenous peptide responses in a pathway-selective manner: implications for drug screening. Mol Pharmacol 78(3): 456-465.

简介

细胞外信号调节激酶1和2(ERK1/2)是在Thr202/Tyr204(ERK1)和Thr185/Tyr187(ERK2)上被磷酸化的丝氨酸/苏氨酸蛋白激酶。 ERK1/2(pERK1/2)的磷酸化来自多个刺激,导致包括细胞生长,增殖和分化的生理反应。 该协议已经优化用于检测表达G蛋白偶联受体(GPCR)的贴壁永生细胞系中配体介导的pERK1/2。

材料和试剂

  1. Dulbecco's改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:11995-065)
  2. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  3. SureFire ®试剂(包括裂解,活化和反应缓冲液)(TGR BioSciences,目录号:TGRES500)
  4. AlphaScreen General IgG(蛋白A)检测试剂盒(PerkinElmer,目录号:6760617)
  5. 白色ProxiPlate 384孔微孔板(PerkinElmer,目录号:6008280)
  6. TopSeal(PerkinElmer,目录号:6005250)
  7. NaCl
  8. KCl
  9. Na HPO 4
  10. KH 2 PO 4
  11. 磷酸盐缓冲盐水(PBS)(见Recipes)
  12. 检测缓冲区(参见配方)

设备

  1. Fusion-α板读数器或Envision板读数器与适当的Alphascreen检测模块(PerkinElmer)
  2. 无菌96孔透明平底板(BD Biosciences,Falcon ,目录号:353072)
  3. 加湿培养箱
  4. 多通道移液器
  5. 微量移液器
  6. 轨道振动器

程序

注意:
1)细胞可以稳定或瞬时表达感兴趣的受体。 2)推荐首先进行时间分析以确定配体介导的pERK1/2最大的时间。 (推荐浓度100×K d)。
    3)然后可以对随后的时间序列进行精制,以确定最大配体诱导的pERK1/2发生的精确时间。


I.   细胞制备

在合适的营养培养基(例如DMEM,10%FBS,无抗生素)中将细胞接种到无菌96孔板中,并在37℃,5%CO 2的潮湿环境中培养, /约为〜90%,第二天融合(约24小时)。

注意:根据所使用的细胞系的细胞数目的优化是必要的(我们建议每孔10,000-50,000个细胞的起始范围)。 CHO FlpIN细胞的推荐密度为30,000个细胞/孔


II。  刺激

  1. 播种后一天,吸出营养培养基,用100μlPBS冲洗一次,并更换为90​​μl预热的DMEM(无FBS)。
  2. 在37℃,5%CO 2的潮湿环境中孵育至少4小时(推荐6小时,直到过夜(O/N))。
  3. 准备在DMEM中的10x终浓度的配体的系列稀释液,对于10μl/孔,以一式两份(最小)进行,并且如果进行时间进程则足够多的时间点。
    注意:使用的浓度范围将取决于配体对受体的亲和力。对于初始时程测试,选择配体和DMEM对照的浓度100×K d。然后可以在随后的实验中改进浓度。如果使用肽或"粘性"配体,在含0.1%BSA的DMEM中制备连续稀释液。
  4. 在DMEM中制备合适浓度的FBS,足以进行10μl/孔,以一式两份(最小)进行,并且如果进行时间进程,足够多的时间点。
    注意:这是实验的内部控制 - FBS促进pERK1/2。 FBS在CHO FlpIN细胞系中的推荐最终浓度为3-10%。
  5. 在DMEM中预孵育后,向细胞中加入10μl10x制备的配体或FBS,总体积为100μl,最终浓度为1x。
    注意:对于初始时间,从90分钟开始,并在每个时间点向细胞添加配体或FBS,直到时间0(无添加)。对于浓度反应,在通过时间过程确定的最大诱导的pERK1/2时添加配体或FBS。
  6. 刺激完成后,从细胞中快速去除含配体的培养基。
    注意:根据细胞类型,这可能涉及轻拂或轻柔吸气。
  7. 加入50μl1x Surefire 裂解缓冲液。
    注意:裂解体积的优化是必要的,并且取决于细胞类型,受体的表达水平和与pERK1/2途径偶联的效率。 推荐在CHO FlpIN细胞系中起始裂解体积为30-100μl。
  8. 孵育裂解物在室温(RT)5-10分钟在轨道摇床


III。 检测

  1. 在减弱的照明条件下,准备检测缓冲液。
  2. 转移5微升细胞裂解液从每个孔到384孔ProxiPlate。
  3. 在减光条件下,向每个样品中加入8.5μl检测缓冲液
  4. 用TopSeal密封板并用箔包裹。
    注意:小体积容易蒸发,TopSeal是至关重要的。
  5. 在减光照条件下室温孵育2小时或37℃孵育1小时。
    注意:如果在37℃下孵育,确保板在测量发光之前回到RT(在37℃温育后约15分钟,在37℃孵育应足够)。 检测珠是温度敏感的。
  6. 使用标准α筛选设置在Fusion-α或Envision读板仪上分析发光。


IV。 数据分析

数据应该归一化到由FBS控制引起的响应。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    137mM NaCl,2.7mM KCl,10mM Na 2 HPO 4,1.8mM KH 2 PO 4,10mM KH 2 PO 4, pH 7.4。
  2. 检测缓冲区
    85.0%SureFire ®反应缓冲液
    14.2%SureFire ®激活缓冲液*
    0.4%受体珠
    0.4%供体珠
    *活化缓冲液应储存在4°C,但在此温度下会发生沉淀。使用前,加热至37°C,以确保所有溶解 在使用前立即准备检测缓冲液。丢弃未使用的检测缓冲区。轻轻混合检测缓冲液。不要涡旋。
    补充说明:裂解物可以储存在-20°C,并且以后检测到pERK1/2,但刺激后不超过2周。

参考文献

  1. Koole,C.,Wootten,D.,Simms,J.,Valant,C.,Sridhar,R.,Woodman,O.L.,Miller,L.J.,Summers,R.J.,Christopoulos,A.and Sexton,P.M。 78(3):456-465。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Koole, C., Wootten, D. and Sexton, P. M. (2013). pERK Detection Assays Using the Surefire AlphaScreen® Kit (TGR Biosciences and PerkinElmer). Bio-protocol 3(13): e806. DOI: 10.21769/BioProtoc.806.
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