Name: assaying protein kinase activity with radiolabeled atp
Uploaded: 2017-05-26T14:00:00
Description: Watch this Scientific Journal Video about Assaying Protein Kinase Activity with Radiolabeled ATP at JoVE.com

作者感谢Cobb实验室的所有现任和前任成员有价值的工作和讨论，Dionne Ware提供行政协助。这些研究得到了National Institutes of Health Grant R37 DK34128和Welch Foundation Grant I1243对MHC的支持

激酶纯化资源和一般免疫沉淀管道注意：用于该测定的激酶可以来源于培养细胞的免疫沉淀物，或通过重组方法如亲和标记的纯化6 。以下是标记免疫沉淀的通用方案，其可能必须根据感兴趣的激酶进行修饰。一切尽可能保持在冰上。 <ol><li>向200μL细胞裂解液（通常〜1 mg / mL总蛋白浓度）中加入2μL1 mg / mL抗体，并在4℃下摇摆孵育1小时。 </li><li> 通过?...

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	<li>Raman, M., Chen, W., Cobb, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+and+properties+of+MAPKs.">Differential regulation and properties of MAPKs.</a> Oncogene. 26 (22), 3100-3112 (2007).</li><li>Wellbrock, C., Arozarena, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Complexity+of+the+ERK/MAP-Kinase+Pathway+and+the+Treatment+of+Melanoma+Skin+Cancer.">The Complexity of the ERK/MAP-Kinase Pathway and the Treatment of Melanoma Skin Cancer.</a> Front Cell Dev Biol. 4, 33 (2016).</li><li>Yang, T., Terman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+PKA-Mediated+Phosphorylation+of+Plexin+Using+Purified+Proteins.">Characterizing PKA-Mediated Phosphorylation of Plexin Using Purified Proteins.</a> Methods Mol Biol. 1493, 147-159 (2017).</li><li>Ross, H., Armstrong, C. G., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+method+for+the+assay+of+many+serine/threonine-specific+protein+kinases.">A non-radioactive method for the assay of many serine/threonine-specific protein kinases.</a> Biochem J. 366 (Pt 3), 977-981 (2002).</li><li>Akita, S., Umezawa, N., Kato, N., Higuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array-based+fluorescence+assay+for+serine/threonine+kinases+using+specific+chemical+reaction.">Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction.</a> Bioorg Med Chem. 16 (16), 7788-7794 (2008).</li><li>Dalby, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Phosphorylation:+Selected+Methods+in+Enzymology.">Protein Phosphorylation: Selected Methods in Enzymology.</a> J Am Chem Soc. 121 (51), 12215 (1999).</li><li>. Autoradiography Available from: <a target="_blank" href="https://www.nationaldiagnostics.com/electrophoresis/article/autoradiography">https://www.nationaldiagnostics.com/electrophoresis/article/autoradiography</a> (2011)</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , (2009).</li><li>Xu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WNK1,+a+novel+mammalian+serine/threonine+protein+kinase+lacking+the+catalytic+lysine+in+subdomain+II.">WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.</a> J Biol Chem. 275 (22), 16795-16801 (2000).</li><li>McReynolds, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+or+Mutation+of+the+ERK2+Activation+Loop+Alters+Oligonucleotide+Binding.">Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding.</a> Biochemistry. 55 (12), 1909-1917 (2016).</li><li>Carlson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+discovery+of+ERK2+substrates+identifies+ERK-mediated+transcriptional+regulation+by+ETV3.">Large-scale discovery of ERK2 substrates identifies ERK-mediated transcriptional regulation by ETV3.</a> Sci Signal. 4 (196), rs11 (2011).</li><li>Courcelles, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+dynamics+reveal+novel+ERK1/2+MAP+kinase+substrates+with+broad+spectrum+of+functions.">Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.</a> Mol Syst Biol. 9, 669 (2013).</li><li>Myers, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Screening+and+Hit+Validation+of+Extracellular-Related+Kinase+5+(ERK5)+Inhibitors.">High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors.</a> ACS Comb Sci. 18 (8), 444-455 (2016).</li></ol>

激酶是一种多样化的蛋白质家族，其在多种情况下已经发展了广泛的功能，激酶测定在研究几种信号蛋白方面已经非常有用，并且极大地有助于我们目前对细胞通信的理解。值得注意的是，尽管在结构和活性上存在重大差异，但使用相同的基本测定来表征两种不同的激酶。 WNK1激酶含有非典型催化口袋，临界赖氨酸转移到独特的位置，已知通过激酶和脚手架功能调节阳离子氯化物共转运蛋白的作?...

蛋白激酶是将细胞信号传递到适当的反应中所必需的复杂酶。鉴于其在维持体内平衡和预防或促进疾病状态方面的作用2 ，用于评估激酶活性的生物化学方法继续是描绘真核信号传导3的特征的有力工具。尽管使用磷酸特异性抗体的策略在测量不同治疗条件对细胞信号传导状态的影响方面已经具有高度的信息，但激酶测定允许直接根据激酶的酶活性来测量不同治疗条件的影响利益。虽然对于不使用放射性物质的类似测定法有几种选择5 ，我们仍然依靠这种方法对结果进行稳健的定量。那里是该测定的两个典型应用，两者都有价值的不同原因：免疫沉淀（IP）激酶测定（ 图1 ）和重组蛋白激酶测定（ 图2 ）。 IP激酶测定对于识别能够激活特异性蛋白激酶的因子以及测量抑制性治疗条件非常有用。简言之，将感兴趣的表位标记的激酶转染到培养的真核细胞中，进行各种处理，免疫沉淀，并测定将放射性标记的磷酸掺入模型底物（ 例如髓磷脂碱性蛋白（MBP））的能力。也可以通过免疫沉淀内源蛋白或任何数量的基因组编辑技术来进行IP激酶测定而不诉诸过表达。因为治疗是在文化中进行的，所以这种方法可以检测通过多个上游因素传播的刺激或通过体外读出的平行通路。该方法的一个主要优点是其不需要在其中直接上游或下游因子或磷酸化位点的先验知识。此外，一旦已经鉴定了感兴趣的激酶的特异性底物，与重组组分可以使用相同的激酶测定方案，以测量与天然底物的比活性，并在与质谱分析结合时鉴定特异性磷酸化位点。使用底物突变体的激酶测定可以进一步验证以这种方式鉴定的位点。最后，该方法也可用于检测和测量自磷酸化。 这里提供的方案假设用于在培养细胞中表达感兴趣的亲和标记的激酶的优化的蛋白质纯化方案或转染方法。有关转染，裂解，免疫沉淀和蛋白质纯化的更详细的论述我们建议参考Cold Spring Harbor Protocols 6 。有关测定开发和修饰的更多信息，请参阅蛋白质磷酸化：选择的酶学方法7 。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein A Sepharose CL-4B</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-0963-03</td>
		</tr>
		<tr>
			<td>Radiolabeled [&#947;-32P] ATP</td>
			<td>Perkin Elmer</td>
			<td>NEG035C010MC</td>
		</tr>
		<tr>
			<td>Laemmli Buffer</td>
			<td>Bio-Rad</td>
			<td>#1610737</td>
		</tr>
		<tr>
			<td>Radioactive shielding</td>
			<td>Research Products International</td>
			<td>BR-006</td>
		</tr>
		<tr>
			<td>R-250 dye (Coomassie)</td>
			<td>Thermo Fisher</td>
			<td>20278</td>
		</tr>
		<tr>
			<td>Whatman Grade 3 qualitative filter paper</td>
			<td>Whatman</td>
			<td>1003-917</td>
		</tr>
		<tr>
			<td>Slab gel vacuum dryer</td>
			<td>Bio-Rad</td>
			<td>Model 583 Gel Dryer #1651745&#160;</td>
		</tr>
		<tr>
			<td>Autorad marker</td>
			<td>Agilent Technologies</td>
			<td>420201</td>
		</tr>
		<tr>
			<td>Phosphorescent ruler</td>
			<td>Sigma Aldrich</td>
			<td>R8133</td>
		</tr>
		<tr>
			<td>Phosphorescent dots</td>
			<td>Sigma Aldrich</td>
			<td>L5149</td>
		</tr>
		<tr>
			<td>Geiger counter</td>
			<td>Ludlum</td>
			<td>Model 3 with 44-9 detector</td>
		</tr>
		<tr>
			<td>BioMax Cassette 8x10&#34;</td>
			<td>Carestream Health</td>
			<td>107 2263</td>
		</tr>
		<tr>
			<td>BioMax MS Film 8x10&#34;</td>
			<td>Carestream Health</td>
			<td>829 4985</td>
		</tr>
		<tr>
			<td>BioMax MS Intensifying Screen 8x10&#34;</td>
			<td>Carestream Health</td>
			<td>851 8706</td>
		</tr>
		<tr>
			<td>Medical/X-ray film processor</td>
			<td>Konica Minolta</td>
			<td>SRX-101A</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Research Products International</td>
			<td>125500</td>
		</tr>
		<tr>
			<td>Scintillation fluid</td>
			<td>MP Biomedicals</td>
			<td>188245305</td>
		</tr>
		<tr>
			<td>Beckman LS 6500 Scintillation counter</td>
			<td>Beckman</td>
			<td>LS 6500</td>
		</tr>
	</tbody>
</table>

assaying protein kinase activity with radiolabeled atp

蛋白激酶能够响应于复杂的刺激阵列来控制大规模细胞变化，并且大量努力旨在揭示其调节的变构细节。激酶包括信号网络，其缺陷通常是多种形式的癌症和相关疾病的标志，使得能够操纵上游调节因子的测定平台和对于改进治疗学的研究至关重要的反应要求的验证。在这里，我们描述了一个基本的激酶测定，可以很容易地适应特定的实验问题，包括但不限于测试生物化学和药理学的作用，遗传操作如突变和缺失，以及细胞培养条件和治疗探针细胞信号传导机制。该测定使用放射性标记的[γ- 32 P] ATP，其允许定量比较和清楚的结果可视化，并且可以是mod如果用于免疫沉淀或重组激酶，特异性或典型的底物，在广泛的反应条件下使用。

WNK1 IP激酶测定将Myc标记的WNK1转染到HEK293细胞中，并用抗Myc抗体11免疫沉淀。免疫沉淀物显示对模型底物MBP以及自身的激酶活性（ 图1A ）。然后通过相同的方法测试WNK1突变体对MBP的激酶活性，此时采用GST标记的构建体（ 图1B ）。相对于野生型的突变体激酶行为的分析揭示了对于最佳WNK...

Watch this Scientific Journal Video about Assaying Protein Kinase Activity with Radiolabeled ATP at JoVE.com

Assaying Protein Kinase Activity with Radiolabeled ATP

蛋白激酶是对于细胞间和细胞内信号转导至关重要的高度进化的信号酶和支架。我们提出了通过使用放射性标记的三磷酸腺苷（[γ- 32 P] ATP）测量激酶活性的方案，这是一种有助于阐明细胞信号调节的可靠方法。

用放射性标记的ATP测定蛋白激酶活性

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

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Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

科研

生物化学

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

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The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

通过对Rho激酶活性的暂时抑制,增强人类诱导多能干细胞衍生心肌细胞的诱导

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...