作者想感谢她的帮助下，在优化这个协议蒂芙尼樱桃。这项工作是由国家衰老研究所（AG032320）以及推进健康威斯康星州。

一个过程的开始与匀浆的基本原理，请参考图1。估计，从细胞中提取的准备钙调素结合蛋白洗脱的时间是6至7小时。 1。组织编制<ol><li>注入病毒包含利益的重组蛋白（在这个例子中，绿色荧光蛋白（GFP）标记伍），并允许组织一夜之间表达蛋白表达质粒器官海马。 </li><li>病毒注射后约12至18小时（取决于病毒表达时间），准备收集组织。加入1ml清扫缓冲区（10MM葡萄糖，4mm的氯化钾，碳酸氢钠26MM，233mM蔗糖，2 5毫米氯化镁，1毫米氯化钙2，和0.1％酚红，供气5％CO 2 95％，O 2的）培养皿。培养组织/插入转移到培养皿中，加入2ml夹层缓冲插入淹没组织。 </li><li>收集吨器官海马组织（5至10片）轻轻刮组织免费使用手术刀插入膜。特别是消除利益的特定区域（如CA1区）也是一种选择。暂停组织转移到1.5ml离心管中，用一个倒置的巴斯德吸管。 </li><li> 1500 RCF 1分钟离心样品分开组织解剖缓冲区。小心取出上清液吸。确保不扰乱沉淀。 </li><li>用于每个切片，加30至60μL同质化的缓冲区（氯化钠150MM，20MM三pH值7.5，1mm的数码地面电视，1μg/mL亮肽素，1μg/mLchemostatin，1μg/mLantipain，1μg/mL胃酶抑素及1％TRITON X -100）组织和同质化彻底与杵。 </li><li>为了去除细胞碎片，离心10分钟在1100 RCF其余匀浆和吸小心取出上清液，同时避免颗粒污染。 </li><li>以10％作为输入的样品（样品1）上清。储存期间准备在第3步使用凸轮琼脂糖珠上冰的剩余上清。 </li></ol>注：这里所用的组织器官的海马。然而，人们可以使用分离的神经元或任何其他的细胞培养系统。在这种情况下，以适当的方式收集您的组织后，开始在步骤1.4。 2。下拉珠的制备在处理珠，重要的是要打捞珠的反应效率，最大限度地防止干燥管双方的珠。要做到这一点，最好是旋转你管，就在自己身边，让解决方案，以湿管的墙壁上的珠子，立即离心前。 <ol><li>对于每个上拉下来，加入400μL悬浮的钙调蛋白琼脂糖凝胶珠成2 ml离心管具有相对FLAT -底部，最大限度地在您的孵化珠您的解决方案的比表面积和互动。 </li><li> 30秒21,000 RCF离心珠，小心取出上清液吸。确保不打扰珠。 </li><li>洗珠，添加100μL含有2个毫米氯化钙2正在使用下拉的Ca 2 + - CAM为珠蛋白和2毫米EDTA（已知的Ca 2 +螯合剂）拉低各自的同质化缓冲区ApoCaM约束力的蛋白质。 1500 RCF 1分钟，轻轻拍打管重悬珠和离心机。小心取出吸上清，确保不打扰珠。 </li></ol>注：对于所有的愿望步骤，建议使用吸头，拥有良好的开放（如凝胶加载提示）允许不拆除的解决方案，消除珠。 3。 CAM -琼脂糖结合的蛋白质<ol><li>拆分成两个同等体积的条件下上清液。根据你的病情，添加适量的氯化钙或EDTA您上清2毫米为每个浓度。 </li><li>从步骤1.7中添加的上清液洗净，在相应的同质化缓冲区的珠子。轻轻拍打管组合。 </li><li>在4 ° C孵育3小时在摇床样品。重新暂停珠每30分钟左右，以增加有约束力的效率。 </li><li>含3分钟1500 RCF离心管样品和珠子。 </li><li>未结合的蛋白质样品（样品2）取上清液50μL，并仔细清除愿望和丢弃剩余的上清。 </li><li>洗珠如在步骤2.3中使用各自的同质化液100μL中所述的三倍。 </li></ol> 4。洗脱<ol><li>加入50μL洗脱缓冲液（50毫米的Tris - HCl pH值为7.5，和150mm氯化钠）含有相反的条件（10MM彗星ACL 2或10mm EDTA）珠。例如，样品匀浆和约束缓冲含Ca 2 +被洗脱洗脱含有EDTA反之亦然的缓冲。 </li></ol>可选：升温洗脱缓冲至37 ° C之前加入珠，可提高产量。 <ol start="2"><li>孵育珠在室温下在摇床30分钟的解决方案。混合轻轻拍打约每5分钟管珠。 </li><li> 3分钟，在1500 RCF离心珠和愿望结合蛋白（样品3）小心取出上清液50μL。确保不打扰珠。 </li><li>所有样品（即匀浆，束缚和结合蛋白，以及那些仍然绑定到珠）加入蛋白上样缓冲液。 </li></ol>注：为了最大限度洗脱（尤其是在效率低下的洗脱的情况下），添加相应的洗脱缓冲液50μL（例如加入EDTA含BUFFER来约束氯化钙2）加热前的样本，以帮助洗脱任何剩余的结合蛋白和4.3中描述的重复步骤，以消除剩余的结合蛋白的珠子珠。 5。 SDS - PAGE和免疫印迹进行SDS - PAGE和分析你的兴趣和探针蛋白探测已知绑定在相反的条件作为阳性对照CAM一种蛋白质，用Western blot。 6。代表性的成果图2B显示一个凸轮下拉检测测试的GFP标记的吴CAM约束力相比，内源性吴的例子。要做到这一点，在我们的器官海马过度表达GFP - NG隔夜和组织匀浆。匀浆孵育与CAM -琼脂糖珠在任的Ca 2 +或EDTA存在。匀浆输入显示，除了内源性吴和Ca 2 + / CaM依赖的蛋白激酶Ⅱ表达GFP - NG（CaMKII）。正如预期的基础上已知的具有约束力的内源性NG（图2a所示），GFP标记吴洗脱的Ca 2 +（EDTA的结合蛋白）的情况下存在的Ca 2 +绑定（图2B） 。相比之下，控制，CaMKII，洗脱的Ca 2 +结合蛋白的存在，并在其缺席（EDTA）的未绑定。这表明，凸轮珠的正常运行和洗脱是有效率的。最重要的，这表明，GFP - NG ApoCaM内源性的形式以类似的方式结合，这表明绿色荧光蛋白标记并没有改变我们的重组蛋白的功能。 <img src="/files/ftp_upload/3502/3502fig1.jpg" alt="图1"/> 图1。CAM下拉试验大纲 （一）组织匀浆降速去除细胞碎片。约10％的上清液作为输入（1）样本。为不同的平分剩余的上清被添加到测试在这些条件的约束条件和适当的试剂（氯化钙或EDTA） 。每个上清（含要么氯化钙或EDTA）装上分别编写的CaM - Sepharose树脂和（二）培养允许绑定。未绑定的蛋白质被删除（2），（三）结合蛋白（3）使用洗脱缓冲液（EB）载有约束力相反的条件珠的洗脱。使用SDS - PAGE和Western blot分析，可以分析这三种蛋白质样品的蛋白质组成。 <img src="/files/ftp_upload/3502/3502fig2.jpg" alt="图2"/> 图2。A.）原理的Ca 2 +依赖的CAM约束力和洗脱下拉检测的例子是两种类型的蛋白质结合在CA 2 CAM +依赖性。神经颗粒素（NG）表示，绑定的apo - CaM和CaMKII代表绑定的Ca 2 +丰富的蛋白质，蛋白质CAM。 CAM是在其分离状态，孵化前的匀浆蛋白。一旦培养高的Ca 2 +浓度（2毫米）的条件下，或存在的CA 2 +螯合剂EDTA（2毫米），蛋白质将绑定到CAM相应。吴在EDTA条件结合CAM，因为几乎没有CA +目前，将洗脱的Ca 2 +的存在凸轮琼脂糖珠。 CaMKII，但是，将绑定到CAM中存在高含量的Ca 2 +，将游离的Ca 2 +螯合后。 B）例如CAM下拉检测结果。这个数字表明，预计最终结果的CAM -琼脂糖拉吴和CaMKII探测与样品。内源性吴和GFP - NG是目前在EDTA存在必然凸轮的蛋白质车道。没有吴是必然样品时，培养与CAM的Ca 2 +的存在，这表明了N克只绑定APO - CAM。另一方面，我们积极的控制，CaMKII，只约束中存在的 Ca 2 CAM + 。

<ol>
	<li>Vincenzi, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+in+the+regulation+of+intracellular+calcium.">Calmodulin in the regulation of intracellular calcium.</a> Proc. West Pharmacol Soc. 22, 289-294 (1979).</li><li>Cheung, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+plays+a+pivotal+role+in+cellular+regulation.">Calmodulin plays a pivotal role in cellular regulation.</a> Science. 207, 19-27 (1980).</li><li>Zhang, M., Tanaka, T., Ikura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-induced+conformational+transition+revealed+by+the+solution+structure+of+apo+calmodulin.">Calcium-induced conformational transition revealed by the solution structure of apo calmodulin.</a> Nat. Struct. Biol. 2, 758-767 (1995).</li><li>Alexander, K. A., Wakim, B. T., Doyle, G. S., Walsh, K. A., Storm, D. R. <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+the+calmodulin-binding+domain+of+neuromodulin,+a+neurospecific+calmodulin-binding+protein.">Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein.</a> J. Biol. Chem. 263, 7544-7549 (1988).</li><li>Huang, K. P., Huang, F. L., Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+7.5-kDa+protein+kinase+C+substrate+(RC3+protein,+neurogranin)+from+rat+brain.">Characterization of a 7.5-kDa protein kinase C substrate (RC3 protein, neurogranin) from rat brain.</a> Arch. Biochem. Biophys. 305, 570-580 (1993).</li><li>Bahler, M., Rhoads, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+signaling+via+the+IQ+motif.">Calmodulin signaling via the IQ motif.</a> FEBS Lett. 513, 107-113 (2002).</li><li>Routtenberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+activation+leading+to+protein+F1+phosphorylation+may+regulate+synaptic+plasticity+by+presynaptic+terminal+growth.">Protein kinase C activation leading to protein F1 phosphorylation may regulate synaptic plasticity by presynaptic terminal growth.</a> Behav. Neural. Biol. 44, 186-200 (1985).</li><li>Zhong, L., Cherry, T., Bies, C. E., Florence, M. A., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+enhances+synaptic+strength+through+its+interaction+with+calmodulin.">Neurogranin enhances synaptic strength through its interaction with calmodulin.</a> EMBO J. 28, 3027-3039 (2009).</li><li>Needham, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+and+adenosinetriphosphate+in+relation+to+muscle+contraction.">Myosin and adenosinetriphosphate in relation to muscle contraction.</a> Biochim. Biophys. Acta. 4, 42-49 (1950).</li><li>Hathaway, D. R., Adelstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+myosin+light+chain+kinase+requires+the+calcium-binding+protein+calmodulin+for+activity.">Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity.</a> Proc. Natl. Acad. Sci. U.S.A. 76, 1653-1657 (1979).</li><li>Fukunaga, K., Yamamoto, H., Matsui, K., Higashi, K., Miyamoto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+a+Ca2+-+and+calmodulin-dependent+protein+kinase+from+rat+brain.">Purification and characterization of a Ca2+- and calmodulin-dependent protein kinase from rat brain.</a> J. Neurochem. 39, 1607-1617 (1982).</li><li>Yang, S. D., Tallant, E. A., Cheung, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin+is+a+calmodulin-dependent+protein+phosphatase.">Calcineurin is a calmodulin-dependent protein phosphatase.</a> Biochem. Biophys. Res. Commun. 106, 1419-1425 (1982).</li><li>Huestis, W. H., Nelson, M. J., Ferrell, J. E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin-dependent+spectrin+kinase+activity+in+human+erythrocytes.">Calmodulin-dependent spectrin kinase activity in human erythrocytes.</a> Prog. Clin. Biol. Res. 56, 137-155 (1981).</li><li>Yamniuk, A. P., Vogel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin's+flexibility+allows+for+promiscuity+in+its+interactions+with+target+proteins+and+peptides.">Calmodulin's flexibility allows for promiscuity in its interactions with target proteins and peptides.</a> Mol. Biotechnol. 27, 33-57 (2004).</li><li>Zhong, L., Kaleka, K. S., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+phosphorylation+fine-tunes+long-term+potentiation.">Neurogranin phosphorylation fine-tunes long-term potentiation.</a> Eur. J. Neurosci. 33, 244-250 (2011).</li></ol>

没有利益冲突的声明。

提供的协议利用CaM的琼脂糖珠调查的Ca 2 +依赖CaM结合蛋白。许多蛋白质结合CAM中的Ca 2 +依赖性。这些相互作​​用CaM结合蛋白的数量和他们的许多信号通路中的关键作用给予高度重视。在这个协议中，CAM - Sepharose树脂用于组织匀浆中的Ca 2 +的存在或缺乏独立于CaM结合蛋白。这个简单的方法的结果将进一步了解蛋白质如何与CAM中的Ca 2 +依赖的方式进行交互。这种方?...

<table border="1"><tr><td> 产品 </td><td> 公司 </td><td> 目录编号 </td><td> 注释 </td></tr><tr><td>钙调蛋白的琼脂糖珠</td><td> GE医疗集团</td><td> 17-0529-01 </td><td></td></tr><tr><td>反CamKII阿尔法</td><td> Sigma - Aldrich公司</td><td> C6974 </td><td></td></tr><tr><td>抗- NG含量</td><td> Millipore公司</td><td> 07-425 </td><td></td></tr><tr><td>凝胶载入中吸取技巧</td><td>费舍尔</td><td> 02-707-138 </td><td>使用上清愿望</td></tr><tr><td>离心管（2.0毫升） </td><td>费舍尔</td><td> 05-408-146 </td><td>使用所有涉及钙调蛋白的琼脂糖珠的步骤</td></tr></table>

pull-down of calmodulin-binding proteins

钙（ 钙离子）离子通过多种机制调节细胞功能的重要。大部分的Ca 2 +信号是通过介导的钙结合蛋白称为钙调蛋白（CAM） 1,2。 CAM是涉及多层次的，在几乎所有的细胞过程，包括细胞凋亡，代谢，平滑肌收缩，突触可塑性，神经生长，炎症和免疫反应。的蛋白质，帮助调节与CAM的互动关系，通过这些途径。许多这些相互作用依赖于CaM的构象，这是明显不同的CA 2 +（的Ca 2 + - CAM）作为反对它的Ca 2 +自由状态（ApoCaM）3时，势必。 虽然大多数靶蛋白结合的Ca 2 + - CAM，某些蛋白质只能绑定到ApoCaM。有的通过他们的智商域绑定CAM，包括neuromodulin的NG（NG）5，4，和一定的肌球蛋白<sup&gt; 6。这些蛋白质已被证实，分别在突触前功能性突触后功能8，和肌肉的收缩9发挥的重要作用。他们的结合能力，并释放钙的缺乏或存在的 CAM +是在其功能的关键。相比之下，许多蛋白质结合的Ca 2 + - CAM，并要求其激活这个约束力。例子包括肌球蛋白轻链激酶10的Ca 2 + / CaM依赖的蛋白激酶（CaMKs）11和磷酸酶（如钙调磷酸酶）12，和血影蛋白激酶13，其中有各种直接和下游的影响14。 这些蛋白质对细胞功能的影响往往依赖于自己的能力结合到CAM中的Ca 2 +依赖性。例如，我们测试的NG - CAM约束力的突触功能的相关性和不同的基因突变如何影响这个约束力。我们生成一个GFP标记的吴CON结构与特定的突变，在IQ域会改变伍CAM中的Ca 2 +依赖性的结合能力。这些不同的基因突变的研究给了我们很大的启示，为参与突触功能的8,15的重要进程。然而，在这些研究中，它是必不可少的证明突变的蛋白质有约束力凸轮预期改变。 在这里，我们目前为检测蛋白质的能力绑定到CAM的Ca 2 +的存在或缺乏，作为例子使用CaMKII和NG方法。这种方法是一种亲和层析的形式简称为CAM的下拉检测。它使用的CaM -琼脂糖珠测试蛋白质结合CAM与此绑定的Ca 2 +的影响。这是相当多的时间效率和需要较少的蛋白质相柱层析及其他实验。总之，这提供了一个宝贵的工具，以探索的Ca 2 + / CaM的信号和蛋白质，在teract与CAM。

Watch this Scientific Journal Video about Pull-down of Calmodulin-binding Proteins at JoVE.com

Pull-down of Calmodulin-binding Proteins

钙调蛋白（CAM）下拉实验调查与各种蛋白质的CaM的相互作用的有效途径。此方法使用效率和具体分析CaM结合蛋白钙调素琼脂糖珠。这提供了一个重要的工具，探索细胞功能的CAM信号。

钙调蛋白结合蛋白的下拉

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Research

JoVE Journal

Biology

25.2K 次观看.  Medical College of Wisconsin. 钙调蛋白（CAM）下拉实验调查与各种蛋白质的CaM的相互作用的有效途径。此方法使用效率和具体分析CaM结合蛋白钙调素琼脂糖珠。这提供了一个重要的工具，探索细胞功能的CAM信号。

视频: Pull-down of Calmodulin-binding Proteins

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

科研

生物学

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

在凝胶染色蛋白质

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

微接触印刷的蛋白细胞生物学

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).