Name: characterization at the molecular level using robust biochemical approaches of a new kinase protein
Uploaded: 2019-06-30T14:00:00
Description: Watch this Scientific Journal Video about Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein at JoVE.com

这项工作得到了癌症协会、L'L'协会神经纤维瘤病和雷克林豪森协会和瓦尔·卢瓦尔共和国中心的支持。非常感谢奥雷利·科松和德博拉·卡萨提供流式细胞学数据,感谢凯龙·希克曼-刘易斯对手稿进行彻底校对。

1. 转染的细胞制备
注意:细胞培养的所有步骤必须在专门的实验室中执行,并且细胞在2类微生物柜内操作。
<ol><li>种子HEK-293(人类胚胎肾)细胞在10 mL的DMEM(Dulbeco的改性鹰中等)的10厘米板中,辅以10%的胎儿小牛血清。在5%CO2下培养3至5天,在37°C下,直到细胞达到+90%的汇合。</li>
	<li>使用胶原蛋白 R 处理 ± 10 厘米板,以增加细胞对塑料板的附着力。<ol>
<li>加入5 mL的胶原蛋白R溶?...

<ol>
	<li>Manning, G., Plowman, G. D., Hunter, T., Sudarsanam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+protein+kinase+signaling+from+yeast+to+man.">Evolution of protein kinase signaling from yeast to man.</a> Trends in Biochemical Sciences. 27, 514-520 (2002).</li><li>Manning, G., Whyte, D. B., Martinez, R., Hunter, T., Sudarsanam, S. <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 kinase complement of the human genome. 298, 1912-1934 (2002).</li><li>Ochoa, D., Bradley, D., Beltrao, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution,+dynamics+and+dysregulation+of+kinase+signalling.">Evolution, dynamics and dysregulation of kinase signalling.</a> Current Opinion in Structural Biology. 48, 133-140 (2018).</li><li>Bhullar, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinase-targeted+cancer+therapies:+progress,+challenges+and+future+directions.">Kinase-targeted cancer therapies: progress, challenges and future directions.</a> Molecular Cancer. 17, 48 (2018).</li><li>Kaboord, B., Perr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+proteins+and+protein+complexes+by+immunoprecipitation.">Isolation of proteins and protein complexes by immunoprecipitation.</a> Methods in Molecular Biology. 424, 349-364 (2008).</li><li>Arnau, J., Lauritzen, C., Petersen, G. E., Pedersen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+strategies+for+the+use+of+affinity+tags+and+tag+removal+for+the+purification+of+recombinant+proteins.">Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins.</a> Protein Expression and Purification. 48, 1-13 (2006).</li><li>Wood, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+trends+and+affinity+tag+designs+for+recombinant+protein+purification.">New trends and affinity tag designs for recombinant protein purification.</a> Current Opinion in Structural Biology. 26, 54-61 (2014).</li><li>Young, C. L., Britton, Z. T., Robinson, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+expression+and+purification:+a+comprehensive+review+of+affinity+tags+and+microbial+applications.">Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications.</a> Biotechnology Journal. 7, 620-634 (2012).</li><li>Greenwood, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+assays+for+sensitive+quantification+of+proteins.">Proximity assays for sensitive quantification of proteins.</a> Biomolecular Detection and Quantification. 4, 10-16 (2015).</li><li>Wang, X., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Dynamic+and+Heterogeneous+Proteasome+Complexes+Using+In+vivo+Cross-Linking-Assisted+Affinity+Purification+and+Mass+Spectrometry.">Dissecting Dynamic and Heterogeneous Proteasome Complexes Using In vivo Cross-Linking-Assisted Affinity Purification and Mass Spectrometry.</a> Methods in Molecular Biology. 1844, 401-410 (2018).</li><li>Raykova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Let+There+Be+Light!.">Let There Be Light!.</a> Proteomes. 4, (2016).</li><li>Weibrecht, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+ligation+assays:+a+recent+addition+to+the+proteomics+toolbox.">Proximity ligation assays: a recent addition to the proteomics toolbox.</a> Expert Review of Proteomics. 7, 401-409 (2010).</li><li>Podobnik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+Study+Protein-protein+Interactions.">How to Study Protein-protein Interactions.</a> Acta Chimica Slovenica. 63, 424-439 (2016).</li><li>Rao, V. S., Srinivas, K., Sujini, G. N., Kumar, G. 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-protein+interaction+detection:+methods+and+analysis.">Protein-protein interaction detection: methods and analysis.</a> International Journal of Proteomics. 2014, 147648 (2014).</li><li>Peck, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+phosphorylation:+methods+and+strategies+for+studying+kinases+and+substrates.">Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates.</a> The Plant Journal: For Cell and Molecular Biology. 45, 512-522 (2006).</li><li>Jia, Y., Quinn, C. M., Kwak, S., Talanian, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+in+vitro+kinase+assay+technologies:+the+quest+for+a+universal+format.">Current in vitro kinase assay technologies: the quest for a universal format.</a> Current Drug Discovery Technologies. 5, 59-69 (2008).</li><li>Okano, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+family+of+serine/threonine+kinases+containing+two+N-terminal+LIM+motifs.">Identification and characterization of a novel family of serine/threonine kinases containing two N-terminal LIM motifs.</a> The Journal of Biological Chemistry. 270, 31321-31330 (1995).</li><li>Croft, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53-mediated+transcriptional+regulation+and+activation+of+the+actin+cytoskeleton.">p53-mediated transcriptional regulation and activation of the actin cytoskeleton.</a> Cell Research. 21, 666-682 (2011).</li><li>Tastet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIMK2-1+is+a+Hominidae-Specific+Isoform+of+LIMK2+Expressed+in+Central+Nervous+System+and+Associated+with+Intellectual+Disability.">LIMK2-1 is a Hominidae-Specific Isoform of LIMK2 Expressed in Central Nervous System and Associated with Intellectual Disability.</a> Neuroscience. 399, 199-210 (2019).</li><li>Vallee, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIMK2-1,+a+new+isoform+of+human+LIMK2,+regulates+actin+cytoskeleton+remodeling+via+a+different+signaling+pathway+than+that+of+its+two+homologs,+LIMK2a+and+LIMK2b.">LIMK2-1, a new isoform of human LIMK2, regulates actin cytoskeleton remodeling via a different signaling pathway than that of its two homologs, LIMK2a and LIMK2b.</a> The Biochemical Journal. 475, 3745-3761 (2018).</li><li>Lee, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Detergents+on+Membrane+Protein+Complex+Isolation.">Impact of Detergents on Membrane Protein Complex Isolation.</a> Journal of Proteome Research. 17, 348-358 (2018).</li></ol>

在这里,我们使用了强大的生化工具,在分子水平上描述一种新的蛋白质LIMK2-1,它被认为是基于其序列和同源性、LIMK2a和LIMK2b20的激酶。
首先,利用西布洛特与特定抗体的分析,在蛋白质水平上证明了LIMK2-1的存在。之后,我们评估了它与上游激酶ROCK1的相互作用,后者已知调节LIMK2a和LIMK2b,LIMK2-1的同源。最后,我们通过体外β32 P+ ATP标签和西布洛特在纤维素中使...

几十年来,许多信号通路已被阐明,并表明它们参与关键的细胞过程,如细胞分裂、分化、运动、程序化细胞死亡、免疫和神经生物学。激酶在这些信号通路中起着重要作用,因为它们经常精细地调节其激活或失活,是响应外部刺激1,2,3的瞬态多功能复合物的一部分。激酶的突变和调节障碍经常导致人类疾病,因此在过去四十年中,它们已成为最重要的药物靶点之一。
在此背景下,能够检测激酶与其上游稳压器或下游基板的相互作用并识别新的合作伙伴非常重要。亲和纯化和免疫沉淀是分离蛋白质复合物5的非常强大的技术。诱饵蛋白或激酶可以标记与特定的肽序列允许使用商业珠子共价结合抗体针对肽。这种材料允许在实验6,7,8中具有很高的可重复性。内源性蛋白也可以使用直接靶饵蛋白的抗体进行免疫沉淀。抗体可能与蛋白A或蛋白GAagagarose珠子交联,或在添加赖沙之前用这些珠子孵育。莱沙缓冲液必须经过优化,使蛋白质溶解而不失去相互作用,并避免蛋白质降解。这种方法的一个主要缺点是相互作用在细胞赖沙时被检测到;因此,瞬态或弱相互作用,以及那些需要亚细胞上下文可能错过。其他技术可用于直接在细胞中工作,如接近结扎测定(PLA)9、体内交联辅助亲亲纯化(XAP)10、生物发光共振能量转移(BRET)或Fürster共振能量转移 (FRET)11,12。此外,免疫沉淀不适合确定结合的热力学常数,需要表面质原共振、等温分滴热量测定或微尺度热泳等物理技术13,14.
可使用多种技术评估激酶活性。在此,我们专注于磷特异性抗体和体外β=32P+ ATP(腺苷三磷酸)标签。磷特异性抗体针对蛋白质中特定残留物的磷酸盐修饰。它们可用于细胞裂解后西布洛特或ELISA(酶链接免疫吸附测定),用于免疫组织化学,以及使用流细胞学或免疫荧光的完整细胞。其缺点可能包括缺乏特异性,可以使用目标蛋白的突变版本进行评估,并且它们不能用于所有蛋白质。体外β=32P+ ATP标签是一种非常健壮、成熟和高度敏感的方法15。可使用免疫沉淀蛋白或重组蛋白,并测试不同的基质。药物的影响也可以评估,因为这种方法是定量的。其主要缺点是,与该方法相关的放射性需要谨慎处理。根据荧光或发光肽基质的测量,利用荧光/发光特性在磷酸化上改变的特性,也可以采用替代方法。这些方法还允许高通量,例如,在筛选可能成为目标激酶潜在抑制剂的分子时,这是必要的。事实上,激酶是制药公司追求的最大药物目标类别之一。
在这项研究中,我们重点研究了LIMK2-1蛋白(LIMK2-1代表Lin11,岛1,Mec3激酶等形2-1)。LIMK2激酶蛋白于1995年首次被描述17。通过替代拼接产生三种 LIMK2 等形:LIMK2a、LIMK2b 和 LIMK2-1。目前,LIMK2-1仅在一项研究18中描述了mRNA水平。在此,我们使用强大的生化方法在分子水平上描述这种潜在的新激酶蛋白。首先,我们证明了LIMK2-1确实是合成的。与LIMK2a和LIMK2b的两种对应物类似,它与上游激酶ROCK(Rho相关蛋白激酶)相互作用。我们显示LIMK2-1在Myelin基本蛋白(MBP)上有激酶活性,但在LIM激酶的规范基质cofilin上没有。

<table>
	<tbody>
		<tr>
			<td>Antibody anti-actin</td>
			<td>Sigma-Aldrich</td>
			<td>A1978</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-c-Myc</td>
			<td>Invitrogen</td>
			<td>MA1-21316</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-cofilin</td>
			<td>Cell signaling Technology</td>
			<td>3312/5175</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-GFP</td>
			<td>Santa Cruz</td>
			<td>sc-9996</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-HA</td>
			<td>Roche Applied Science</td>
			<td>11687423001</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-phospho-cofilin</td>
			<td>Cell signaling Technology</td>
			<td>3313</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody Anti-PP1i</td>
			<td>Eurogentec</td>
			<td>designed for this study</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Euromedex</td>
			<td>A-162B</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Invitrogen</td>
			<td>PV3227</td>
			<td>for g[32P] labeling</td>
		</tr>
		<tr>
			<td>g[32P] ATP</td>
			<td>Perkin Elmer</td>
			<td>NEG502A</td>
			<td>for g[32P] labeling</td>
		</tr>
		<tr>
			<td>BES buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>14280</td>
			<td>for transfection</td>
		</tr>
		<tr>
			<td>b-glycerophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>b-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td>for Laemmli</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td>for blocking buffer</td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126</td>
			<td>for Laemmli</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td>for transfection</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td>111-541</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen R</td>
			<td>Pan Biotech</td>
			<td>P06-20166</td>
			<td>for transfection</td>
		</tr>
		<tr>
			<td>Control siRNA</td>
			<td>Ambion</td>
			<td>AM4611</td>
			<td>for PP1i antibody specificity</td>
		</tr>
		<tr>
			<td>Coomassie PageBlue Protein Staining Solution</td>
			<td>Thermo-Fisher</td>
			<td>24620</td>
			<td>for gel staining</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>3690</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>Electrophoresis Unit</td>
			<td>Biorad</td>
			<td>Mini-Protean</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>EZview Red anti-HA affinity gel</td>
			<td>Sigma-Aldrich</td>
			<td>E6779</td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr>
			<td>GeneSys software</td>
			<td>Ozyme</td>
			<td></td>
			<td>for Western Blot acquisition</td>
		</tr>
		<tr>
			<td>GeneTolls software</td>
			<td>Ozyme</td>
			<td></td>
			<td>for Western Blot quantification</td>
		</tr>
		<tr>
			<td>GFP-trap beads</td>
			<td>Chromtek</td>
			<td></td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Euromedex</td>
			<td>26-128-6405</td>
			<td>for transfer buffer</td>
		</tr>
		<tr>
			<td>GST-cofilin</td>
			<td>Upstate Cell signaling</td>
			<td>12-556</td>
			<td>for g[32P] labeling</td>
		</tr>
		<tr>
			<td>Hamilton syringe 100 mL</td>
			<td>Hamilton</td>
			<td>710</td>
			<td>to remove carefully supernatant from beads without aspirating them</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>for kinase buffer</td>
		</tr>
		<tr>
			<td>ImageQuant TL software</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>for radioactivity acquisition and quantification</td>
		</tr>
		<tr>
			<td>LIMK2 siRNA</td>
			<td>Ambion</td>
			<td>s8191</td>
			<td>for PP1i antibody specificity</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma-Aldrich</td>
			<td>SP-04-2217</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>MBP</td>
			<td>Upstate Cell signaling</td>
			<td>13-173</td>
			<td>for g[32P] labeling</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>for kinase buffer</td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>244589</td>
			<td>for kinase buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Euromedex</td>
			<td>1112</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Sigma-Aldrich</td>
			<td>S-1504</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>Okaidic acid</td>
			<td>Euromedex</td>
			<td>0-2220</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>78830</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>p-nitrophenylphosphate</td>
			<td>Euromedex</td>
			<td>1026</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>PVDF membrane Immobillon-P</td>
			<td>Merck-Millipore</td>
			<td>IPVH00010 pore size 0,45 mm</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Rotating wheel</td>
			<td>Labinco</td>
			<td></td>
			<td>for bead incubation</td>
		</tr>
		<tr>
			<td>Safe lock eppendorf</td>
			<td>Eppendorf</td>
			<td>0030120.086</td>
			<td>for kinase assay</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>5030</td>
			<td>for Laemmli and migration buffer</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>LC Laboratories</td>
			<td>S8507</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate</td>
			<td>Fluka</td>
			<td>71501</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>Super Signal West Dura</td>
			<td>Protein Biology</td>
			<td>34075</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Syngene Pxi</td>
			<td>Ozyme</td>
			<td></td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Tissue extracts 
			 
			&#160;</td>
			<td>Biochain 
			 
			&#160;</td>
			<td>P1234035 Brain 
			P12345152 Lung 
			P1234149 Liver 
			P1234188 Pancreas 
			P1234260 Testis</td>
			<td>for Western Blot analysis 
			 
			&#160;</td>
		</tr>
		<tr>
			<td>Transfer Unit</td>
			<td>Biorad</td>
			<td>Mini-Trans-Blot</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Euromedex</td>
			<td>26-128-3094 B</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949</td>
			<td>for blocking buffer</td>
		</tr>
		<tr>
			<td>Typhoon FLA9500</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>to read autoradiography</td>
		</tr>
		<tr>
			<td>Typhoon Trio</td>
			<td>Amersham Bioscience</td>
			<td></td>
			<td>to read autoradiography</td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>GE Healthcare</td>
			<td>3030-672</td>
			<td>for Western Blot</td>
		</tr>
	</tbody>
</table>

characterization at the molecular level using robust biochemical approaches of a new kinase protein

广泛的全基因组测序已经确定了许多开放阅读框架(ORFs),提供了许多潜在的蛋白质。这些蛋白质可能对细胞有重要作用,并可能解开新的细胞过程。在蛋白质中,激酶是主要参与者,因为它们属于细胞信号通路,能够打开或关闭许多对细胞命运至关重要的过程,如细胞生长、分裂、分化、运动和死亡。
在这项研究中,我们重点研究了一种新的潜在激酶蛋白LIMK2-1。我们用特定的抗体证明了它的存在。我们使用共免疫沉淀实验评估其与上游调节蛋白的相互作用。共免疫沉淀是一种非常强大的技术,能够检测两个目标蛋白之间的相互作用。它也可以用来检测诱饵蛋白的新伙伴。诱饵蛋白可以通过设计到其序列的标签进行纯化,或者通过专门针对它的抗体进行纯化。然后,这些蛋白质复合物可以通过SDS-PAGE(硫酸二苯基苯甲酸钠聚丙烯酰胺凝胶)分离,并使用质谱法进行鉴定。免疫沉淀LIMK2-1也用于通过β+32P+ ATP标签在体外测试其激酶活性。这种成熟的测定可能使用许多不同的基质,诱饵的突变版本可用于评估特定残留物的作用。药理剂的作用也可以评估,因为这种技术是高度敏感和定量的。尽管如此,放射性处理需要特别小心。激酶活性也可以评估与特定抗体针对改性氨基酸的磷组。这些类型的抗体并非适用于所有磷改性残留物。

合成LIMK2-1蛋白 LIMK2-1在数据库中被提及,但到目前为止只有一篇论文表明其mRNA18的存在。与LIMK2a和LIMK2b的两个同源体相比,LIMK2-1有一个额外的C端域,被识别为蛋白磷酸酶1抑制域(PP1i)。我们设计了一种针对该领域的肽的抗体,氨基酸671-684(图1A)。 针对人类蛋白质数据库的BLAST研究表明,只有一种蛋白质,PHI-1(磷酸酶全酶抑制...

Watch this Scientific Journal Video about Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein at JoVE.com

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

我们使用强大的生化方法对一种新的激酶蛋白进行了定性:用特殊抗体对不同细胞系和组织进行西带体分析,通过共免疫沉淀实验相互作用,由西方检测到的激酶活性使用磷特异性抗体和由 β+32P+ ATP 标记的斑点。

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 ...

免疫沉淀是一种非常强大的技术，用于分离和纯化靶蛋白。在平滑条件下，蛋白质、调节器或基质可以共同免疫沉淀。然后，可能会发现一个新的交互网络。免疫沉淀蛋白的活性也可以评估。特别是，激酶的活性可以通过P32-ATP标签在不同基材上进行测试。然而，针对与疾病有关激酶的抑制剂的活性可以进行评估。它们可能构成开发新药的铅化合物。这种方法可以从哺乳动物扩展到酵母或细菌。这种技术并不难。要点：确保具有负控制，以确保检测到的交互是特定的，并仔细选择解解条件以保持相互作用和活动。小细节和手势对于确保这项技术的成功非常重要，它们永远不会在文章的材料和方法中描述。演示程序将是法比安戈丁，一个技术员从我们的实验室。在生物安全柜内的细胞培养室中准备细胞后，使用10毫升移液器将介质从盘子中去除，并丢弃在带漂白剂的专用废物瓶中。然后，加入10毫升新鲜补充的DMEM，并在37摄氏度时将盘子放回孵化器。为了准备转染混合物，在15毫升管中，在pH 7.5下加入450微升10毫摩尔三氧化盐溶液，辅以1毫摩尔EDTA和50微升2.5摩尔氯化钙溶液。通过反转混合。进入管中，加入一个对应于10微克血浆DNA的体积，由DNA中滴答包扩增，并稀释与该试剂盒的洗脱缓冲液，其浓度已确定。反转管子混合。然后，在涡流搅拌器上平滑搅拌，缓慢地加入500微升的 BES 缓冲盐水2X浓缩物，一滴一滴地滴入管中。小心移动，不要干扰DNA和磷酸钙之间的复杂形成。在室温下孵育至少15分钟，或长达45分钟。现在，将板从培养箱转移到安全柜。将准备好的DNA复合物一滴一滴地添加到盘子表面的细胞上。在37摄氏度的孵化器中孵育24至72小时。为了防止蛋白质降解，在冰上准备解液缓冲液，考虑每个转染板的四毫升解液缓冲液。从培养箱中取出带转染细胞的盘子。取出介质并将其丢弃在专用漂白剂废瓶中。要清洗细胞，在板的一侧添加三毫升的冷PBS滴滴，以避免分离转染细胞。轻轻旋转板，将缓冲液铺到表面上。倾斜板以取出 PBS 并将其丢弃到废瓶中。重复一次洗涤。将盘子放在冰上。最后，小心地拆下 PBS 的其余部分。接下来，在转染洗涤细胞中加入500微升的冷解缓冲液。摊在盘子表面。在冰上孵育10分钟。不时地将缓冲液铺在板面上。之后，使用细胞铲刮掉表面的细胞，并在微离心管中收集。在10，000倍的G和4摄氏度下进行10分钟的离心机。将上流液酸盐收集到新的微离心管中，并收集该分数的 50 微升等分，再收集另一个新的微离心管。此等分物将用于检查转染蛋白是否表达良好。将颗粒丢弃在垃圾桶中。首先，轻轻重新暂停与适当抗体相结合的阿加罗斯珠的库存溶液。切开 200 微升尖端的端，使开口 1 到 2 毫米。将尖端连接到微管，并绘制 40 微升溶液，让珠子进入尖端。向上和向下移液珠数次，使珠子饱和，以确保珠子的正确体积，并转移珠子到微离心管。加入500微升TNET缓冲液，通过反转混合，在1000倍的G和4摄氏度下离心两分钟。小心地取出上流液，并添加 500 微升的 TNET 缓冲液。在旋转轮上孵育至少一小时，温度为4摄氏度。然后，在1000倍的G和4摄氏度下将管子离心两分钟，然后用500微升的解液缓冲液通过倒置和离心两次清洗珠子。洗涤后，将先前收集的总馏分将酸盐放入管中，并在四摄氏度的旋转轮上孵育两到四个小时。现在，在1000倍的G和4摄氏度下，将含有免疫沉淀珠的管子离心两分钟。通过倒置和离心，用500微升的解解缓冲液洗涤珠子五次。清洗珠子时，注意不要离珠子太近，同时去除上一液。否则，珠子将被吸气，在实验结束时，将没有更多的珠子。对于洗脱，第一台离心机在1000度G和4摄氏度下进行两分钟的离心机。使用一毫升微管小心地取出上一液。然后，用装有水泥针的汉密尔顿注射器，取出上一滴，以避免珠子吸入。接下来，添加 40 微升 4X Laemmli 缓冲液。轻轻敲击管子，使其均匀。在室温下孵育五分钟。然后，在10，000倍的G和室温下进行5分钟的离心机。使用汉密尔顿注射器，将上流水液转移到新的微离心管中。储存在零下80摄氏度。HEK-293 细胞被未标记的 LIMK 2-1 或 LIMK 2 的 HA 标记等构形式之一转染。LIMK 2-1 在不同的条件下表达良好。抗 LIMK 2-1 抗体不识别 LIMK 2a 和 LIMK 2b 等构形式，并且是特异性的，因为与控制小干扰RNA相比，当细胞与 LIMK2 小干扰RNA进行转染时，其信号会降低。在各种人类细胞系中，LIMK 2-1似乎在 HEK-293 和 HeLa 中表达，但在 C6 中不表达。利用共生沉淀实验评估LIMK2-1与上游激酶岩石的相互作用。通过转染载体编码的每个不同的蛋白质都表达得很好。LIMK2 的三个等同形式以及幼虫 6 被有效免疫沉淀。在水线中，检测到 ROCK，因此与 LIMK2 的三个等构形式共生，但与 larp6 不一起沉淀。然后检测到的交互是特定的。通过伽玛P32 ATP标签测试了共生素LIMK 2-1的激酶活性。LIMK 2-1没有磷酸酯共生素，而它做磷酸化米林基本蛋白，或MBP。LIMK 2-1 在此测试中有效免疫沉淀。在进行免疫沉淀时进行负控制至关重要，以确保相互作用是特定的。必须仔细设置解液缓冲液的组成，以保持相互作用和活动。在免疫沉淀时，可评估激酶活性以计算不同的基质。突变可能很容易引入，并可能解开特定残留物的作用。也可以进行功能研究，以了解新的突出相互作用的生理作用。细胞必须在生物安全柜内的专用培养室中培养。P32 ATP 必须谨慎处理。防护罩可防止辐射、盖革计数器、特定废物收集、个人乳房和手指徽章，以检测放射性暴露和过滤提示。

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Assaying Protein Kinase Activity with Radiolabeled ATP

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 ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

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

人类外周血神经细胞分离通过评估Rab10磷酸化来询问帕金森氏症相关的LRRK2激酶途径

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 ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

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. ...