This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), the Takeda Science Foundation (HT), and by a Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) to HT.

1.转<ol><li>种子在100毫米培养皿（2×10 6个细胞/皿）HEK293细胞。培养在补充有10％胎牛血清，100μg/ ml的链霉素和100单位/ ml青霉素的细胞在Dulbecco改进的Eagle氏培养基。孵育在加湿培养箱将细胞在5％CO 2和37℃。 </li><li>第二天，转染细胞10微克FLAG标记ARIP4质粒按照制造商的指示11，用40微升转染试剂用。 </li></ol> 2.蛋白提取<ol><li>约36小时转染后，立即用冰冷的PBS将细胞并使用刮刀收获细胞。转移细胞到1.5 mltube并在4℃下以8000 xg离心离心它为2分钟。吸出上清，重悬细胞沉淀在5×0.4米氯化钾缓冲的沉淀量（高盐缓冲液:20毫摩尔的Tris-HCl（pH为8.0），0.4M氯化钾，5mM的MgCl 2的，10％甘油，0.1％NP-40，10毫B-巯基乙醇，1×蛋白酶抑制剂鸡尾酒）。旋转该混合物在4℃下20分钟。 </li><li>离心管中，在17700 XG在4℃下10分钟。 </li><li>上清液（全细胞提取物）转移到新的2毫升管中，并添加3×0米氯化钾缓冲的初始体积（稀释缓冲液:20毫摩尔的Tris-HCl（pH值8.0），5mM的MgCl 2的，10％甘油， 0.1％的NP-40，10毫β巯基乙醇，1×蛋白酶抑制剂鸡尾酒）。 </li><li>执行另一个离心（17700×g离心4℃10分钟），上清液（全细胞提取物）转移到新的2ml管中。 </li></ol> 3.免疫沉淀<ol><li>在离心（2.4），洗50微升用PBS抗FLAG珠，0.1M甘氨酸 - 盐酸盐，然后的1M Tris盐酸，然后洗涤两次，用0.1M氯化钾缓冲液（低盐缓冲液:20毫摩尔三 - 盐酸（pH值8.0），0.1M氯化钾，5mM的氯化镁，10％甘油，0.1％NP-40，10μMM B巯基乙醇，1×蛋白酶抑制剂鸡尾酒）。此洗涤过程中离心分离是在2000 xg离心在4℃下进行1分钟。 </li><li>混合50微升FLAG珠与全细胞提取物，并在2-4℃（从步骤2.4全细胞提取物的1％也应保持在-80℃以供将来使用轻轻旋转混合物4小时为输入）。 </li><li>样品转移到微离心柱（重力下降）。保持流通在1.5ml管中，利用液氮冷冻，然后将其存储在-80℃下（检查输入和用Western印迹分析的流通的蛋白水平）。 </li><li>加入1 ml 0.1米氯化钾缓冲液（低盐缓冲液）的洗FLAG珠（重力下降）。 </li><li>重复步骤3.4至少四次（对总共五个洗涤）。 </li></ol> 4.洗脱<ol><li>放置柱到1.5毫升管，并在2000 xg离心离心它为1分钟;珠会干。 </li><li>盖的底列。 </li><li>添加50微升（等于FLAG珠的体积）0.1M甘氨酸 - 盐酸（pH值2.5）的。 </li><li>轻轻晃动，在室温下10分钟的混合物。 </li><li>放置柱到一个新的1.5毫升管，并在2000 xg离心离心它为1分钟。 </li><li>洗脱溶液与10微升的1M的Tris-HCl（pH 8.0）中的中和;通过移液或者通过使用涡旋机拌匀。 </li></ol> 5.烷基化和胰蛋白酶消化<ol><li>添加100mM的NH 4 HCO 3，10微升CH 3 CN的，和2μl的二硫苏糖醇的混合物（112微升总体积）的50微升。 </li><li>在56℃孵育样品30分钟。 </li><li>放置在室温下的样品10分钟。 </li><li>添加10微升333毫摩尔的碘乙酰胺的样品，并在37°C孵育它在黑暗中30分钟。 </li><li>添加胰蛋白酶的10微升（33纳克/微升）到混合物中，并在37℃过夜孵育它。 </li></ol><ol><li>以下过夜胰蛋白酶，运到最终产品的LC-MS / MS设施或用于分析12第三方质谱实验室。 </li></ol>

<ol>
	<li>De Las Rivas, J., Fontanillo, C. <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+interactions+essentials:+key+concepts+to+building+and+analyzing+interactome+networks.">Protein-protein interactions essentials: key concepts to building and analyzing interactome networks.</a> PLoS Comput.Biol. 6 (6), e1000807 (2010).</li><li>Westermarck, J., Ivaska, J., Corthals, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+interactions+involved+in+cellular+signaling.">Identification of protein interactions involved in cellular signaling.</a> Mol.Cell.Proteomics. 12 (7), 1752-1763 (2013).</li><li>MacPherson, R. E., Ramos, S. V., Vandenboom, R., Roy, B. D., Peters, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+PLIN+proteins,+ATGL+and+CGI-58,+interactions+at+rest+and+following+stimulated+contraction.">Skeletal muscle PLIN proteins, ATGL and CGI-58, interactions at rest and following stimulated contraction.</a> Am.J.Physiol.Regul.Integr.Comp.Physiol. 304 (8), 644-650 (2013).</li><li>Qin, K., Dong, C., Wu, G., Lambert, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactive-state+preassembly+of+G(q)-coupled+receptors+and+G(q)+heterotrimers.">Inactive-state preassembly of G(q)-coupled receptors and G(q) heterotrimers.</a> Nat.Chem.Biol. 7 (10), 740-747 (2011).</li><li>Ogawa, H., Ishiguro, K., Gaubatz, S., Livingston, D. M., Nakatani, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+with+chromatin+modifiers+that+occupies+E2F-+and+Myc-responsive+genes+in+G0+cells.">A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells.</a> Science. 296 (5570), 1132-1136 (2002).</li><li>Shi, Y., 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=Coordinated+histone+modifications+mediated+by+a+CtBP+co-repressor+complex.">Coordinated histone modifications mediated by a CtBP co-repressor complex.</a> Nature. 422 (6933), 735-738 (2003).</li><li>Huh, K. W., DeMasi, J., Ogawa, H., Nakatani, Y., Howley, P. M., Munger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+human+papillomavirus+type+16+E7+oncoprotein+with+the+600-kDa+retinoblastoma+protein-associated+factor,+p600.">Association of the human papillomavirus type 16 E7 oncoprotein with the 600-kDa retinoblastoma protein-associated factor, p600.</a> Proc.Natl.Acad.Sci.U.S.A. 102 (32), 11492-11497 (2005).</li><li>Huang, B. X., Kim, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+identification+of+Akt+interacting+proteins+by+two-step+chemical+crosslinking,+co-immunoprecipitation+and+mass+spectrometry.">Effective identification of Akt interacting proteins by two-step chemical crosslinking, co-immunoprecipitation and mass spectrometry.</a> PLoS One. 8 (4), 61430 (2013).</li><li>ten Have, S., Boulon, S., Ahmad, Y., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+immuno-precipitation+proteomics+-+the+user's+guide.">Mass spectrometry-based immuno-precipitation proteomics - the user's guide.</a> Proteomics. 11 (6), 1153-1159 (2011).</li><li>Ogawa, H., Komatsu, T., Hiraoka, Y., Morohashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Suppression+by+Transient+Recruitment+of+ARIP4+to+Sumoylated+nuclear+receptor+Ad4BP/SF-1.">Transcriptional Suppression by Transient Recruitment of ARIP4 to Sumoylated nuclear receptor Ad4BP/SF-1.</a> Mol.Biol.Cell. 20 (19), 4235-4245 (2009).</li><li>Tsuchiya, 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=Selective+autophagic+receptor+p62+regulates+the+abundance+of+transcriptional+coregulator+ARIP4+during+nutrient+starvation.">Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation.</a> Sci.Rep. 5, 14498 (2015).</li><li>Watanabe, T., Matsuo, I., Maruyama, J., Kitamoto, K., Ito, Y. <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+an+intracellular+lectin,+calnexin,+from+Aspergillus+oryzae+using+N-glycan-conjugated+beads.">Identification and characterization of an intracellular lectin, calnexin, from Aspergillus oryzae using N-glycan-conjugated beads.</a> Biosci.Biotechnol.Biochem. 71 (11), 2688-2696 (2007).</li><li>Sitz, J. H., Tigges, M., Baumgartel, K., Khaspekov, L. G., Lutz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dyrk1A+potentiates+steroid+hormone-induced+transcription+via+the+chromatin+remodeling+factor+Arip4.">Dyrk1A potentiates steroid hormone-induced transcription via the chromatin remodeling factor Arip4.</a> Mol.Cell.Biol. 24 (13), 5821-5834 (2004).</li><li>Mohammed, H., Taylor, C., Brown, G. D., Papachristou, E. K., Carroll, J. S., D'Santos, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+immunoprecipitation+mass+spectrometry+of+endogenous+proteins+(RIME)+for+analysis+of+chromatin+complexes.">Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes.</a> Nat.Protoc. 11 (2), 316-326 (2016).</li><li>Fonslow, B. R., Yates, 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=Capillary+electrophoresis+applied+to+proteomic+analysis.">Capillary electrophoresis applied to proteomic analysis.</a> J.Sep.Sci. 32 (8), 1175-1188 (2009).</li></ol>

The authors have nothing to disclose.

有效转染是必不可少的获得与该协议的成功的结果。因此，我们建议您使用Western blot分析，以确定免疫沉淀的FLAG标签的蛋白质水平。这个步骤允许用户以验证他们感兴趣的蛋白被正确过度表达，此外，该IP被成功执行。 FLAG标签的蛋白质水平也应之前的质谱分析进行检查。 有模拟样本应该被用作阴性对照，以避免得到假阳性结果，并以鉴别从真相互作用的背景。此外，研究人类蛋白质时，实验应在相对干净的环境（ 例如，洁净室）中进行，以避免污染。虽然具有高盐浓度（0.3-0.4 M）的蛋白质提取缓冲液可被偶尔用于提取核蛋白，具有0.1-0.15 1M盐的浓度的IP缓冲液是大多数十大推荐。这使得IP步骤而不破坏蛋白质 - 蛋白质相互作用进行。为了进一步减少背景信号，磁珠可以用于纯化过程。同样重要的是，优化了细胞裂解条件，以降低假阳性结果。 在这项研究中相对于其它技术所描述的方法的显着性（基于消化例如，在凝胶单一蛋白识别）5，10，11是它允许用户为高通量鉴定进行LC-MS / MS分析核辅助因子及其结合合作伙伴之间的蛋白相互作用。转录的基因调控是高度依赖于序列特异性DNA结合蛋白的合作。因此，转录因子通常具有大量的辅因子相互作用，以改变靶基因的表达。钍<html> erefore，以更好地理解这些管理机制转录，确定促进靶蛋白的活性是最重要的转录合作伙伴的细胞活动。我们的方法允许用户通过他们感兴趣的转录因子快速识别假定核辅助因子，并进一步加强了我们的转录调控机制的认识。 FLAG肽也可被用来代替0.1M甘氨酸 - 盐酸溶液（pH 2.5）中洗脱过程。然而，如果采用此变形例中，用于稀释FLAG肽的缓冲液的pH应被监控。 3X FLAG肽也应该用于从FLAG珠洗脱蛋白。因此，具有相对高的pH值的溶液，应使用以稀释这些修饰的条件下，FLAG肽。它也至关重要，以避免以保持适当的蛋白质 - 蛋白质相互作用的样品的反复冻融。 t"的&gt;有时，胰蛋白酶消化过程的优化，可能还需要用于提高蛋白质相互作用筛选结果的准确性。虽然在LC-MS / MS法是可以用来识别核之间的相互作用的工具蛋白质，它的局限性之一是，它不提供关于该蛋白质复合物的装配准确的信息。为了提高复合体稳定性，化学交联处理可以使用14。作为另一种方法，毛细管电泳-质谱/质谱也是有用的15 。

蛋白质 - 蛋白质相互作用发挥多种生物学功能中起重要作用。正因为如此，这些相互作用已牵涉信号转导;跨细胞膜蛋白质运输;细胞的新陈代谢;和几个核过程，包括DNA复制，DNA损伤修复，重组和转录1，2，3，4。查明参与这些相互作用蛋白质因此，推进我们的这些细胞过程的理解是至关重要的。 免疫沉淀（IP）是用于分析蛋白质 - 蛋白质相互作用经过验证的技术。为了便于共同免疫沉淀的蛋白的鉴定，质谱常常利用5，6，7，8，9。通过用抗体靶向的蛋白复合物的公知的部件，它是可以分离的蛋白复合物，并随后经由质谱分析确定其未知组分。 ARIP4（雄激素受体相互作用蛋白4），一个转录coregulator，核受体蛋白以激活或在一个依赖于上下文的方式9,10抑制其靶启动子相互作用。为了更好地了解控制这些核因子的转录调控机制，我们描述了一个全面的方法来净化和使用LC-MS / MS系统识别ARIP4相互作用的蛋白质。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:71px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11668019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Protease Inhibitor Cocktail (EDTA free) (100x)</td>
			<td style="width:71px;">nacalai tesque</td>
			<td style="width:119px;">03969-21</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">ANTI-FLAG M2 Affinity Gel</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Micro Bio-Spin Columns</td>
			<td style="width:71px;">BIO-RAD</td>
			<td style="width:119px;">732-6204</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protein preparation method for the high-throughput identification of proteins interacting with a nuclear cofactor using lc-ms/ms analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

我们确定了一个强烈的信号ARIP4，约160千道尔顿，以及那些一个模拟采样控制等几个未知的蛋白质（ 图1）。 LC-MS / MS分析，鉴定两ARIP4复杂肽和FLAG珠（ 表1）的级分中的潜在的肽辅因子。 P62（Sequestosome1），公知的辅因子ARIP4 11中的分析（ 表1），这证实了本系统11的功效被确定。我们还确定了与ARIP4互动其它许多以往报道的蛋白质。以这种方式，SUMO2 10和DryK 13经由LC-MS / MS分析进行检测。 ARIP4肽使用LC-MS / MS分析，这表明该IP是高品质也确定。总体而言，LC-MS / MS技术是可以用于识别未知核事件链接的强大工具编到蛋白质 - 蛋白质相互作用。全面质量分析结果将根据要求提供。 <img alt="图1" src="/files/ftp_upload/55077/55077fig1.jpg" /> 图1:ARIP4配合物的净化。 FLAG表位标记的ARIP4的银染（ARIP4-F），在HEK293细胞和免疫沉淀表达与特定FLAG抗体。纯化的蛋白用凝胶电泳分离并用银染色显示。作为对照，对HEK293细胞不表达外源蛋白质，进行一个模拟纯化。分子量标准显示在左侧。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55077/55077fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/55077/55077table1.jpg" /> 表1:辨识新型ARIP4结合蛋白氮。结合到纯化的蛋白质复合物FLAG珠通过基于胰蛋白酶消化解离。用LC-MS / MS分析这些蛋白质随后鉴定。使用SWISS-PROT数据库和MASCOT软件在LC-MS / MS数据进行分析。肽的数量和这些蛋白质的身份被示出。只选择与显著MASCOT评分（P &lt;0.05）的蛋白质。

Watch this Scientific Journal Video about A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis at JoVE.com

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

我们已经建立了使用LC-MS / MS系统共同调节相互作用蛋白的纯化的方法。

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

a-protein-preparation-method-for-high-throughput-identification

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.2K 次观看.  Osaka University. 我们已经建立了使用LC-MS / MS系统共同调节相互作用蛋白的纯化的方法。 

视频: A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

高通量, 用定量斑点杂交分析法绝对测定组织层中选定蛋白质的含量 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

从脂质绑定蛋白和过敏原中去除和替换内源性利根

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

使用多柱板适配器的经济性和多功能性高通量蛋白质净化系统

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...