搜索

IP-Kinase Assay
免疫沉淀激酶活性检测试验   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Immunoprecipitation (IP)- Kinase assays are an invaluable tool to assess the activation status of intracellular signaling cascades within a specific cellular state and also to confirm the enzymatic activity of a specific kinase towards a putative substrate of interest. Intracellular signal transduction cascades play an important role in modulating the localization of transcription factors and thus impact the cellular transcriptome. This in turn regulates key cell fate decisions including cell survival, apoptosis, proliferation, and differentiation. Here we describe an in vitro non-radioactive method to assess kinase activity towards a specific substrate. In this protocol we outline the method for Akt, however the basic protocol may be applied to any kinase and putative substrate of interest.

Keywords: Pluripotency(多能性), Kinase(激酶), In-vitro assay(体外实验), Akt(Akt), Stem cells(干细胞)

Materials and Reagents

  1. Recombinantly produced substrate of interest
    For this study, full-length murine Oct4 was cloned into a mammalian expression vector containing a T3 promoter sequence and a carboxy-terminal 6X His – TEV – 3X FLAG epitope tage.
  2. TnT Coupled Reticulocyte Lysate Systems (Promega Corporation, catalog number: L5010 )
  3. Expression vector (with Sp6, T3, or T7 promoter sequence) containing protein of interest (Test Substrate) wild-type, putative mutant, empty vector control. You will also need a vector containing a previously confirmed (and published) target substrate if one is not commercially available to use as a control for the kinase assay.
  4. Transcend tRNA (Promega Corporation, catalog number: L5061 )
  5. RNaseOUT (Life Technologies, catalog number: 10777019 )
  6. Cell line which exhibits activity for kinase of interest
  7. SDS polyacrylamide gel
  8. Polyvinyl difluoride membrane (PVDF) (Bio-Rad Laboratories, catalog number: 162-0177 )
  9. Primary Antibody to Test and Control Substrates
    1. Akt (total) (Cell Signaling Technology, catalog number: 4685 )
    2. Phospho-Akt Substrate Antibody (Cell Signaling Technology, catalog number: 10001 )
    3. Gsk3 (total) (Cell Signaling Technology, catalog number: 5676 )
  10. Kinase agonist (if required) to augment kinase activity
    This protocol uses Ro-31-8220 (Sigma-Aldrich, catalog number: R136-5MG ) as an Akt agonist
  11. Non-radioactive Akt Kinase Assay Kit (Cell Signaling Technology, catalog number: 9840 )
    1. Immobilized Phospho-Akt (Ser473) (D9E) Rabbit mAb (Bead Conjugate)
    2. Phospho-GSK-3 (Ser21/9) (37F11) Rabbit mAb
    3. GSK-3 Fusion Protein at 0.5 mg/ml
    4. 10 mM ATP (50 µl)
  12. Bead conjugated primary antibody or primary antibody and Protein A/G Agarose (Pierce Antibodies)
  13. Phenyl methlsulfonyl fluoride (PMSF) (Sigma-Aldrich)
  14. 1x Cell Lysis Buffer (see Recipes)
  15. 1x Kinase Buffer (see Recipes)
  16. 3x SDS Sample Buffer (see Recipes)

Equipment

  1. Mini-Western/Transfer Apparatus (Bio-Rad Laboratories)
  2. 10 cm plates
  3. Cell scraper
  4. 1.5 ml microfuge tube
  5. Refrigerated microfuge
  6. Microfuge tube rotator
  7. Heat blocks set to 30 and 95 °C

Procedure

  1. In vitro transcription/translation
    1. Prepare the recombinantly produced substrate of interest using the TnT Coupled Reticulocyte Lysate System. Wild-type, kinase mutant, and empty vector control will be required.
    2. Following the manufacturer’s recommended protocol proceed to set up the following reaction mixes for each sample. All reagents should be kept on ice until step C3.

      Component
      Volume (µl)
      TnT Lysate
      25
      TnT Reaction Buffer
      2
      T3 RNA Polymerase*
      1
      Amino Acids-Met
      0.5
      Amino Acids-Leu
      0.5
      RNaseOUT
      1
      Transcend tRNA
      1
      H2O
      14
      DNA (0.2 µg/µl for 1 µg total)
      5
      *This kit requires that the protein of interest be cloned in an expression vector containing an SP6, T3, or T7 promoter sequence. The appropriate polymerase must be selected based on vector utilized.

    3. Incubate the reactions assembled in Step A2 for 90 min at 30 °C.
    4. Typical yields from coupled transcription/translation are from 50-500 ng/ µl.
    5. Analyze the product by Western blot. Combine 5 µl of the reaction with 20 µl of 1x SDS loading buffer. Denature at 95 °C for 3 min and load 10 µl onto an SDS polyacrylamide gel. Transfer gel to PVDF membrane. Detect using a primary antibody raised against the protein of interest and/or epitope tag contained in the selected vector to confirm expression.
    6. Store the remaining in vitro transcription/translation reaction at -20 °C until further use in step D.

  2. Cell lysate preparation
    1. Culture actively growing 10T1/2 Fibroblasts to 75% confluence. Four 10 cm plates will be sufficient for assay of control, wild-type, and putative Akt mutant substrates.
    2. Culture the cells in the presence of 10 µM Ro-31-8220 at 37 °C for one hour to increase Akt activation.
    3. Aspirate media and quickly rinse the cells twice with ice cold PBS, aspirating between each wash.
    4. Lyse cells with complete 1x Cell Lysis Buffer supplemented with 1 mM PMSF. Use 0.5 ml per 10 cm plate. Incubate on ice for 5 min.
    5. Remove cells from plate with cell scraper. Place lysate in 1.5 ml microfuge tube on ice for 30 min gently vortexing two times (setting 6) for 10 sec each at 10 and 20 min. Sonication is not necessary.
    6. Centrifuge the lysate at 10,000 x g for 10 min at 4 °C. Transfer the supernatant to a fresh tube. Store lysate at -80 °C until use.

  3. Immunoprecipitation
    1. Each experiment will require eight immunoprecipitations; four with cell lysate and four mock immunoprecipitations with 1x Cell Lysis Buffer. For each set of four immunoprecipitations, one will be a positive control for kinase activity, employing a previously confirmed (in the literature) substrate. The remaining three will be used for the recombinantly produced test substrate (wild-type, putative kinase mutant, and empty vector). The mock immunoprecipitation is a negative control used to ensure that the kinase activity emanates from the cell lysate and not other reagents used during this protocol.
    2. Add 20 µl of immobilized antibody-bead slurry to 200 µl of lysate (or 1x Cell Lysis Buffer) in a 1.5 ml microfuge tube. Incubate overnight at 4 °C with end-over-end rotation. Proceed to step C6. Immobilized phospho-Akt (Ser473) is included in the Non-Radioactive Akt-kinase Assay Kit.
    3. Alternatively, add primary antibody to 200 µl of lysate. The exact amount may need to be titrated. Approximately 1 µg of an affinity-purified antibody is generally sufficient.
    4. Incubate overnight at 4 °C with end-over-end rotation.
    5. Add prepared Protein A/G Agarose beads (25 µl of 50% slurry). Incubate at 4 °C with end-over-end rotation for 2 h.
    6. Centrifuge the immunoprecipitate at 10,000 x g for 30 sec at 4 °C. Aspirate off supernatant and wash the pellet (on ice) two times for 3 min each with 500 µl 1x Cell Lysis Buffer.
    7. Wash the pellet (on ice) two times for 3 min with 500 µl 1x Kinase Buffer.

  4. On-bead non-radioactive in vitro kinase assay
    1. Resuspend the final pellet in 50 µl of 1x Kinase Buffer.
    2. Add 1 µl of 10 mM ATP (from the Non-radioactive Akt Kinase Assay Kit) and 1 or 2 µl of kinase substrate generated in step A6.
    3. Incubate for 30 min at 30 °C.
    4. To terminate the reaction add 25 µl of 3x SDS Sample Buffer. Vortex gently, then microfuge at 10,000 x g to collect.
    5. Store at -80 °C or proceed directly to analyze by Western.

  5. Western analysis
    1. Heat each required sample at 95 °C for 3 min.
      1. Properly controlled experiments should contain Westerns showing:
        1. Kinase (total and activated form) in the absence and presence of agonist to confirm that the kinase is active.
        2. Control IP-kinase assays using a previously confirmed kinase substrate. Duplicate Westerns for the mock and kinase exposed samples should be run for incubation with antibodies directed to both the total and phosphorylated form of the protein.
        3. Test IP-kinase assays showing empty vector, wild-type, and putative kinase mutant form of the substrate of interest for mock and kinase exposed samples. Duplicate Westerns should be run as above. If an antibody directed to the phosphorylated form of the protein of interest is not commercially available, a phospho-kinase substrate antibody may be used in its place since it will only detect substrates when they are phosphorylated.

  6. Interpretation
    1. A putative substrate is confirmed when:
      1. Kinase activity is confirmed in step E1a.i.
      2. Phosphorylation of the control substrate is confirmed in step E1a.ii kinase exposed sample, but not in the mock exposed sample.
      3. Phosphorylation of putative substrate is confirmed in wild-type but not the putative kinase mutant form of the protein. No expression of total protein or phospho-protein should be observed for the empty vector control.

Recipes

  1. 1x Cell Lysis Buffer
    25 mM Tris (pH 7.5)
    150 mM NaCl
    1 mM EDTA
    1 mM EGTA
    1% Triton
    2.5 mM Na4P2O7
    1 mM β-Glycerophosphate
    1 mM Na3VO4
    1 µg/µl Leupeptin
    Stored at 4 °C for 1-2 weeks only
  2. 1x Kinase Buffer
    25 mM Tris (pH 7.5)
    5 mM β-Glycerophosphate
    2 mM DTT
    0.1 mM Na3VO4
    10 mM MgCl2
    Stored at -20 °C
    May be stored at 4 °C for 1-2 weeks only
  3. 3x SDS Sample Buffer
    187.5 mM Tris (pH 6.8)
    6% w/v SDS
    30% glycerol
    150 mM DTT
    0.03% w/v bromophenol blue
    Aliquot and stored at -20 °C
    Add DTT fresh before each use

Acknowledgments

This work was supported by grants from Genome Canada, the Ontario Genomics Institute, the Stem Cell Network, and the Canadian Institutes of Health Research.

References

  1. Campbell, P. A. and Rudnicki, M. A. (2013). Oct4 interaction with Hmgb2 regulates Akt signaling and pluripotency. Stem Cells 31(6): 1107-1120.

简介

免疫沉淀(IP) - 激酶测定是评估特定细胞状态内的细胞内信号级联的活化状态以及确认特异性激酶朝向目的基因的酶活性的有价值的工具。 细胞内信号转导级联在调节转录因子的定位中发挥重要作用,从而影响细胞转录组。 这反过来调节关键细胞命运决定,包括细胞存活,凋亡,增殖和分化。 在这里,我们描述了一种体外非放射性方法来评估对特定底物的激酶活性。 在这个协议中,我们概述了Akt的方法,但是基本协议可以应用于任何激酶和感兴趣的推定底物。

关键字:多能性, 激酶, 体外实验, Akt, 干细胞

材料和试剂

  1. 重组产生的感兴趣底物
    对于该研究,将全长鼠Oct4克隆到含有T3启动子序列和羧基末端6X His-TEV-3X FLAG表位的哺乳动物表达载体中。
  2. TnT偶联网状细胞溶解物系统(Promega Corporation,目录号:L5010)
  3. 含有感兴趣的蛋白质(测试底物)野生型,推定的突变体,空载体对照的表达载体(具有Sp6,T3或T7启动子序列)。 如果不能用作激酶测定的对照,则还需要含有先前确认(和公布的)靶底物的载体。
  4. Transcend tRNA(Promega Corporation,目录号:L5061)
  5. RNaseOUT(Life Technologies,目录号:10777019)
  6. 对感兴趣的激酶显示活性的细胞系
  7. SDS聚丙烯酰胺凝胶
  8. 聚二氟乙烯膜(PVDF)(Bio-Rad Laboratories,目录号:162-0177)
  9. 用于测试和控制底物的初级抗体
    1. Akt(总计)(Cell Signaling Technology,目录号:4685)
    2. Phospho-Akt底物抗体(Cell Signaling Technology,目录号:10001)
    3. Gsk3(总计)(Cell Signaling Technology,目录号:5676)
  10. 激酶激动剂(如果需要)以增强激酶活性
    该方案使用Ro-31-8220(Sigma-Aldrich,目录号:R136-5MG)作为Akt激动剂
  11. 非放射性Akt激酶测定试剂盒(Cell Signaling Technology,目录号:9840)
    1. 固定化磷酸-Akt(Ser473)(D9E)兔mAb(珠缀合物)
    2. 磷酸-GSK-3(Ser21/9)(37F11)兔mAb
    3. GSK-3融合蛋白,浓度为0.5mg/ml
    4. 10mM ATP(50μl)
  12. 珠连接的一抗或一抗和蛋白A/G琼脂糖(Pierce抗体)
  13. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich)
  14. 1x细胞裂解缓冲液(参见配方)
  15. 1×激酶缓冲液(参见配方)
  16. 3x SDS样品缓冲液(参见配方)

设备

  1. Mini-Western/Transfer Apparatus(Bio-Rad Laboratories)
  2. 10厘米板
  3. 细胞刮刀
  4. 1.5 ml微量离心管
  5. 冷冻微离心器
  6. 微量离心管旋转器
  7. 加热块设置为30和95℃

程序

  1. 体外转录/翻译
    1. 使用TnT耦合网织红细胞裂解物系统制备重组产生的感兴趣的底物。 将需要野生型,激酶突变体和空载体对照
    2. 按照制造商推荐的方案进行,为每个样品设置以下反应混合物。 所有试剂应保存在冰上,直到步骤C3
      组件
      体积(μl)
      TnT裂解液
      25
      TnT反应缓冲液
      2
      T3 RNA聚合酶*
      1
      氨基酸-met
      0.5
      氨基酸 - 亮氨酸
      0.5
      RNaseOUT
      1
      Transcend tRNA
      1
      H sub 2 O
      14
      DNA(0.2μg/μl,总计1μg)
      5
      该试剂盒需要将目的蛋白克隆到含有SP6,T3或T7启动子序列的表达载体中。 必须根据所使用的载体选择合适的聚合酶。

    3. 在30℃下孵育步骤A2中组装的反应90分钟
    4. 偶联转录/翻译的典型产率为50-500ng /μl
    5. 通过蛋白质印迹分析产物。 将5μl的反应与20μl的1x SDS上样缓冲液混合。 在95℃变性3分钟,并加载10μl到SDS聚丙烯酰胺凝胶上。 转移凝胶到PVDF膜。 使用针对所选载体中包含的目的蛋白质和/或表位标签产生的一抗来检测以确认表达
    6. 将剩余的体外转录/翻译反应储存在-20℃下,直到在步骤D中进一步使用
  2. 细胞裂解液制备
    1. 文化积极生长10T1/2成纤维细胞至75%汇合。 四个10cm板将足以用于对照,野生型和推定的Akt突变体底物的测定
    2. 在10μMRo-31-8220的存在下在37℃下培养细胞1小时以增加Akt活化。
    3. 吸出培养基,并用冰冷的PBS快速冲洗细胞两次,每次洗涤之间抽吸
    4. 用完全1x细胞裂解缓冲液补充1mM PMSF裂解细胞。 使用0.5毫升每10厘米板。 在冰上孵育5分钟。
    5. 用细胞刮刀从板上取下细胞。 将裂解物置于1.5ml微量离心管中冰上30分钟,在10和20分钟,轻轻涡旋两次(设置6)10秒。 不需要声处理。
    6. 在4℃下以10,000×g离心裂解物10分钟。 转移上清液到一个新鲜的管。 将裂解物储存在-80℃直至使用
  3. 免疫沉淀
    1. 每个实验将需要八次免疫沉淀;四个细胞裂解物和四个模拟免疫沉淀与1x细胞裂解缓冲液。对于每组四种免疫沉淀,使用先前证实的(在文献中)底物,一个是激酶活性的阳性对照。剩余的三个将用于重组产生的测试底物(野生型,推定的激酶突变体和空载体)。模拟免疫沉淀是用于确保激酶活性从细胞裂解物中发出的阴性对照,而不是在该方案期间使用的其它试剂。
    2. 加入20微升的固定化抗体珠浆液到200微升裂解液(或1x细胞裂解缓冲液)在1.5毫升微量离心管。在4℃下,在末端旋转下孵育过夜。进行到步骤C6。固定的磷酸-Akt(Ser473)包括在非放射性Akt-激酶测定试剂盒中。
    3. 或者,向200μl裂解物中加入一抗。确切的量可能需要滴定。大约1μg的亲和纯化的抗体通常就足够了
    4. 在4℃下孵育过夜,并进行端到端旋转
    5. 加入准备好的蛋白A/G琼脂糖珠(25μl的50%浆液)。 在4℃下孵育2小时,结束 - 旋转。
    6. 在4℃下以10,000×g离心免疫沉淀30秒。 吸出上清液,用500μl1x细胞裂解缓冲液洗涤沉淀(在冰上)两次,每次3分钟。
    7. 用500μl1x激酶缓冲液洗涤沉淀(在冰上)两次,每次3分钟
  4. 在珠上非放射性的体外激酶测定
    1. 重悬在50μl的1×激酶缓冲液中的最终沉淀。
    2. 加入1μl10 mM ATP(来自非放射性Akt激酶测定试剂盒)和1或2μl步骤A6中产生的激酶底物。
    3. 在30℃下孵育30分钟。
    4. 终止反应,加入25μl3×SDS样品缓冲液。 轻轻涡旋,然后以10,000×g 微量离心收集
    5. 存储在-80°C或直接进行西方分析
  5. 西方分析
    1. 将每个所需样品在95℃加热3分钟。
      1. 适当控制的实验应包括西方显示:
        1. 在不存在和存在激动剂的情况下激酶(总和活化形式),以确认激酶是活性的
        2. 使用先前确认的激酶底物的对照IP-激酶测定。对于模拟和激酶暴露的样品的重复Western应该与针对蛋白质的总的和磷酸化形式的抗体一起孵育。
        3. 测试IP-激酶测定显示模拟和激酶暴露的样品的感兴趣底物的空载体,野生型和推定的激酶突变体形式。重复的西方应该如上运行。如果针对目标蛋白质的磷酸化形式的抗体不是商业上可获得的,则磷酸激酶底物抗体可以用于其位置,因为它将仅在它们被磷酸化时检测底物。

  6. 解释
    1. 当以下情况时确认假定的底物:
      1. 在步骤E1a.i中确认激酶活性
      2. 在步骤E1a.ii激酶暴露的样品中,但不在模拟暴露的样品中,确认对照底物的磷酸化。
      3. 在野生型中证实推定底物的磷酸化,但不是蛋白质的推定的激酶突变体形式。 对于空载体对照,不应观察到总蛋白或磷酸蛋白的表达

食谱

  1. 1x细胞裂解缓冲液
    25mM Tris(pH7.5) 150mM NaCl 1mM EDTA
    1 mM EGTA
    1%Triton
    2.5mM Na 4 P 2 O 7 sub 1 mMβ-磷酸甘油 1mM Na 3 VO 4 sub。
    1μg/μl亮肽素 在4°C下储存1-2周只有
  2. 1×激酶缓冲液
    25mM Tris(pH7.5) 5 mMβ-磷酸甘油 2mM DTT
    0.1mM Na 3+ VO 4
    10mM MgCl 2/
    储存于-20°C
    可能在4°C储存1-2周仅有
  3. 3x SDS样品缓冲液
    187.5mM Tris(pH6.8) 6%w/v SDS
    30%甘油 150 mM DTT
    0.03%w/v溴酚蓝
    等分并储存在-20°C
    在每次使用前添加DTT

致谢

这项工作得到了来自加拿大基因组,安大略基因组研究所,干细胞网络和加拿大健康研究所的资助。

参考文献

  1. Campbell,P.A。和Rudnicki,M.A。(2013)。 Oct4与Hmgb2的相互作用调节Akt信号转导和多能性。干细胞 31(6):1107-1120。
  • 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: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Campbell, P. A. (2014). IP-Kinase Assay. Bio-protocol 4(5): e1059. DOI: 10.21769/BioProtoc.1059.
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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。