我们感谢慷慨支持的几个基金会和慈善捐赠（见SakmarLab.org）。

1。非天然氨基酸位点特异性基因掺入到G蛋白偶联受体<ol><li>保持HEK293T细胞在DMEM（4.5 g / L的葡萄糖，2mM谷氨酰胺）中在37℃下在5％CO 2气氛中添加了10％胎牛血清（FBS）。 </li><li>转染生长至在使用Lipofectamine试剂加有10厘米板60-80％汇合的细胞。 <ol><li>到750微升DMEM中，加入10微升加试剂，3.5微克的GPCR的cDNA（pMT4.的Rho或染pcDNA3.1。CCR5）含有琥珀终止密码子在所希望的位置上，3.5微克的抑制基因tRNA的cDNA（pSVB.Yam）和0.35微克的突变氨酰tRNA合成酶的cDNA 的 p-叠氮基-L-苯丙氨酸（pcDNA.RS）。在室温下孵育15分钟。也可以执行使用重量GPCR的cDNA（不含有琥珀终止密码子），以用作对照类似转染。将此混合物750μLDMEM与17μL脂质体。平衡15分钟后在室温下，使总体积〜4毫升。 </li><li>吸出介质上10厘米的板，采用转染混合物到细胞中，并返回到37℃，在5％CO 2气氛中。后4-6小时，补充细胞用4ml的DMEM含20％FBS和1 mM的AZF。 </li></ol></li><li>第二天，用含有10％FBS和0.5mM AZF DMEM更换生长培养基。 </li><li>收获细胞48小时后转染，分析表达或继续在下面的章节中描述光交联或标签的程序。 <ol><li>确认在UAA的生长培养基中存在的G蛋白偶联受体琥珀突变体的表达。我们通过免疫印迹检测。溶胞物，以1％的细胞沉淀物（重量/体积）十二烷基-β-D-麦芽糖苷（DDM）在20mM的Tris-HCl，pH值7.5，100mM NaCl的1小时，在4℃下离心溶胞产物以16,000×g离心并解决下通过SDS-PAGE在还原条件上清液。转移到PVDF膜上，并进行免疫印迹分析使用日检测全长受体的表达Ë1D4单抗（国家细胞培养中心），其中确认在受体的C-末端的工程表位。31 </li></ol></li></ol> 2。映射一个配体结合网站使用定向光交<ol><li> 48小时转染后，从细胞中吸出介质，并替换为4毫升1X磷酸盐缓冲盐水（PBS）中。返回到37°C下15分钟。 </li><li>重悬细胞用1×PBS和转移到Falcon管中。离心机在1500 rpm离心2分钟以沉淀细胞并吸出上清液。 </li><li>重悬含有20mM HEPES，pH为7.5，0.2％BSA和氚化的配体在饱和浓度的结合的时间和温度所需要的长度，以确保受体 - 配体复合物形成的细胞沉淀物在1×Hank氏缓冲盐溶液。 </li><li>细胞悬液转移到24孔或96孔板的AZF的光活化在365nm下在4℃下（ 例如，使用UV-A灯）15分钟Maintain中板置于冰上，以避免样品加热和细胞裂解。 </li><li>将细胞转移到Eppendorf管中，离心沉淀细胞，除去上清液并进行分析。这些颗粒可以被储存在-20℃下以供将来使用。 </li><li>重悬含1％（重量/体积）的DDM，20mM的Tris-盐酸，pH为7.5和蛋白酶抑制剂的细胞沉淀中的裂解缓冲液。经过1小时温育在4℃下，离心分离细胞裂解物对于以16,000×g离心10分钟，并且将上清液转移到新管中。 </li><li>添加1D4单克隆抗体 - 琼脂糖树脂2B向上清液孵育过夜，在4℃使用改造的C-末端1D4单克隆抗体的表位至immunopurify的G蛋白偶联受体。第二天，用裂解缓冲液洗珠数次。洗脱的样品用1％SDS在37℃下进行1小时振荡培养。 </li><li>洗脱剂的一部分转移到15ml小瓶中含闪烁液并计数在β闪烁计数器来量化氚标记的配体的特异性结合量以日Ë受体。 </li><li>解决剩余1D4单克隆抗体纯化洗脱液用标准凝胶电泳。我们使用了4-12％SDS-PAGE凝胶，然后半干转移到PVDF膜用于免疫印迹分析。我们还包括了与标准蛋白质阶梯车道引导分子量的视觉逼近。 <ol><li>用含有0.05％Tween-20的5％牛奶的TBS缓冲液阻断PVDF膜上。探测膜，以确认全长受体表达与1D4单克隆抗体随后HRP-偶联的抗小鼠IgG第二抗体。 </li><li>治疗用增强的化学发光（ECL）试剂和暴露膜放射自显影胶片，以可视化的频带。 </li></ol></li><li>切膜用刀片来分隔每个样品泳道，接着通过切割在特定分子量标记物。转移膜段含闪烁液小瓶。通过计数β-闪烁计数器定量在每个膜段氚的量。 </li><li>标识正面photocrosslink与氚的表观分子量等于该G蛋白偶联受体和配体的总和的检测。 </li></ol> 3。有针对性的抗原表位标记和细胞表面酶联免疫吸附试验（ELISA） <ol><li> 24小时转染后，洗涤细胞，用1ml的1×PBS和胰蛋白酶消化，用1ml 0.25％胰蛋白酶3分钟。补充用9ml的DMEM含有10％FBS和0.5mM AZF。重悬细胞并计数细胞密度。我们使用标准的血球。 </li><li>预治疗的高结合，透明底的96孔板中，用1​​00ml的0.01毫克/毫升，每孔聚-D-赖氨酸。温育30分钟，在室温下用1×PBS和风干反复洗涤。 </li><li>板200微升转染细胞在6×10 4的密度- 8×10 4个细胞/孔的96孔板中，并返回到37℃，在5％CO 2气氛中。 </li><li>第二天准备的细胞进行标记。轻轻洗涤三次，用100μl/孔的1×PBS吨Ø删除任何AZF含有生长培养基。确保细胞仍附着，例如，通过使用PBS中含有的Ca 2 +和Mg 2 +。 </li><li>从维持在5-20毫米PBS股票准备50-200微米旗 - 三芳基膦标记试剂在1x HBSS / PBS中。申请60毫升到每个孔中（一式三份对各琥珀突变体），并返回到37℃，进行30分钟至4小时。保持一个集水井没有在1x HBSS / PBS标签处理。还包括G蛋白偶联受体重量控制AZF-G蛋白偶联受体的变体来比较。 </li><li>用100μl/孔的封闭缓冲液洗涤细胞3次，BB（1×PBS，0.5％BSA），以完全除去未反应的标记。 </li><li>应用100μl/孔的4％多聚甲醛（从在1x PBS中的16％的库存制备）到细胞中。孵育20分钟，在室温下进行。带BB洗三次。 </li><li>执行标准细胞表面的ELISA方法检测标记的受体和受体的表达。孵育一抗1.5小时冰上其次是次要的tibody在室温下孵育1小时。 <ol><li>加入100μl抗FLAG M2抗体（ 如抗体1:2000稀释的BB制造）检测FLAG-三芳基膦标记试剂的FLAG肽抗原表位。还探讨非标签处理的孔作为阴性对照。添加抗CCR5 2D7（我们用1:500稀释）抗体，以一个单独的集水井，以确定重量或AZF-GPCR细胞表面表达。 </li><li>带BB洗涤细胞3次，并孵育用100μlHRP-偶联的抗小鼠IgG第二抗体（1:2000稀释）的。 </li><li>小心地用BB洗涤细胞五次。加入50μL检测缓冲液：红色的Amplex，20 mM的H 2 O 2，1×PBS（1:10:90的比例），并孵育15分钟，在室温下。收集的荧光多孔板读数器的光谱数据在λEX = 530和λ 发射 = 590。 </li></ol></li></ol> 4。一个G蛋白偶联受体的生物正交荧光标记<ol><li>裂解10 7收获的细胞中表达（重量）或AZF-视紫红质中含有1％1毫升溶解缓冲液变体（重量/体积）DM，50mM的HEPES或Tris-盐酸，pH为6.8，100 mM氯化钠，1mM的氯化钙和蛋白酶抑制剂为1小时，在4℃下离心细胞裂解物在15,000×g离心10分钟，收集上清液部分。 </li><li>加入100μl1D4-单克隆抗体琼脂糖凝胶2B树脂使用设计的C末端1D4表位捕捉受体。孵育12小时，在4℃下用0.1％的洗涤树脂3次（重量/体积）的DDM在0.1M磷酸钠缓冲液，pH 7.3。 </li><li>加入0.1毫米的荧光素膦0.3总体积 - 0.5毫升到标签受体固定在免疫亲和基质（1D4-单抗琼脂糖2B）。孵育12小时，在室温下进行。离心机除去上清液。洗涤树脂以除去未反应的标签。 </li><li>洗脱标记的受体与含0.1％（重量/体积）的DDM和0.33毫克/毫升的九肽（C9对1D4表位肽）100毫升洗脱缓冲液在2 mM磷酸盐缓冲液，pH 6.0通过在冰上温育1小时。 </li><li>在还原条件下解决标记的样品进行SDS-PAGE。在短暂的PBS洗凝胶，然后通过凝胶内的荧光可视化AZF-G蛋白偶联受体的标签。例如，使用共聚焦荧光扫描仪具有488纳米波长的激光来检测受体修饰与荧光素。 </li></ol>

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J. 84, 2078-2100 (2004).</li><li>Okada, T., 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=The+retinal+conformation+and+its+environment+in+rhodopsin+in+light+of+a+new+2.2+&#197;+crystal+structure.">The retinal conformation and its environment in rhodopsin in light of a new 2.2 &#197; crystal structure.</a> J. Mol. Biol. 342, 571-583 (2004).</li><li>Huber, T., Tian, H., Ye, S., Naganathan, S., Kazmi, M. A., Duarte, T., Sakmar, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+labeling+of+functional+G+protein-coupled+receptors+at+genetically+encoded+azido+groups.">Bioorthogonal labeling of functional G protein-coupled receptors at genetically encoded azido groups.</a> , (2013).</li><li>Naganathan, S., Ye, S., Sakmar, T. P., Huber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+epitope+tagging+of+GPCRs+by+bioorthogonal+modification+of+a+genetically-encoded+unnatural+amino+acid.">Site-specific epitope tagging of GPCRs by bioorthogonal modification of a genetically-encoded unnatural amino acid.</a> Biochemistry. , (2013).</li><li>Molday, R. S., MacKenzie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+rhodopsin:+characterization,+cross-reactivity,+and+application+as+structural+probes.">Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes.</a> Biochemistry. 22, 653-660 (1983).</li><li>Gether, U., 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=Agonists+induce+conformational+changes+in+transmembrane+domains+III+and+VI+of+the+beta2+adrenoceptor.">Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.</a> The EMBO journal. 16, 6737-6747 (1997).</li><li>Hubbell, W. L., Altenbach, C., Hubbell, C. M., Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+structure,+dynamics,+and+activation:+A+perspective+from+crystallography,+site-directed+spin+labeling,+sulfhydryl+reactivity,+and+disulfide+cross-linking.">Rhodopsin structure, dynamics, and activation: A perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking.</a> Advances in Protein Chemistry; Membrane Proteins. 63, 243-290 (2003).</li><li>Cai, K., Itoh, Y., Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+contact+sites+in+complex+formation+between+transducin+and+light-activated+rhodopsin+by+covalent+crosslinking:+use+of+a+photoactivatable+reagent.">Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 4877-4882 (2001).</li><li>Dorman, G., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzophenone+photophores+in+biochemistry.">Benzophenone photophores in biochemistry.</a> Biochemistry. 33, 5661-5673 (1994).</li><li>Zhang, Z., 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=A+new+strategy+for+the+site-specific+modification+of+proteins+in+vivo.">A new strategy for the site-specific modification of proteins in vivo.</a> Biochemistry. 42, 6735-6746 (2003).</li><li>Dirksen, A., Hackeng, T. M., Dawson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophilic+catalysis+of+oxime+ligation.">Nucleophilic catalysis of oxime ligation.</a> Angew. Chem. Int. Ed. 45, 7581-7584 (2006).</li><li>Yanagisawa, T., 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=Multistep+engineering+of+pyrrolysyl-tRNA+synthetase+to+genetically+encode+N(epsilon)-(o-azidobenzyloxycarbonyl)+lysine+for+site-specific+protein+modification.">Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification.</a> Chemistry &amp; biology. 15, 1187-1197 (2008).</li></ol>

作者什么都没有透露。

在这里，我们描述了一个完善的方法进行位点特异性结合反应的探针，AZF，进入G蛋白偶联受体，并证明这个工具来研究G蛋白偶联受体的结构和动力学的三个有用的应用程序。我们的方法来位点特异性地并入UAAs规避与基于化学标记32,33或附加光交联剂34到单个可访问的半胱氨酸突变体的替代策略的一个基本问题。虽然，针对半胱氨酸硫醇基团的化学已被用于附加的荧光探针，这种?...

七螺旋G蛋白偶联受体（GPCR）包括高度动态的膜蛋白介导的重要和多样的细胞外信号的超家族。在古典范式，受体激活耦接配体诱导的构象变化。1，在G蛋白偶联受体的结构生物学2最近的进步已经提供的跨膜信号转导的分子机制显著洞察力。3-5然而，为了理解具有更大的化学精度的作用机理和G蛋白偶联受体信号传导的结构动力学，办法的工具包，需要把信息分子和化学探针审问积极信号复合体。 为此，我们适应的方法来位点特异性地引入基于非天然氨基酸（UAA）的诱变使用琥珀密码子之前由舒尔茨和c开创抑制技术非或最低限度地扰动探测到表达的受体oworkers 6我们优化了UAA诱变的方法来实现对蛋白质，如G蛋白偶联受体，它们是难以表达，在大多数的异源系统比哺乳动物细胞等的高产量表达和诱变系统。使用正交设计的抑制性tRNA和进化氨酰-tRNA合成酶对用于特定UAA，我们位点特异性地引入UAAs中表达靶GPCR的。 UAAs的成功的掺入， 对 -乙酰基-L-苯丙氨酸（ACF）， 对 -苯甲酰基-L-苯丙氨酸（BZF），和p-叠氮基-L-苯丙氨酸（AZF）已被证明在我们的模型中的GPCR -视紫红质和人类CC趋化因子受体，CCR5 7,8 原则上，UAA可以基因编码的蛋白质序列中的任何位置，这个属性是一个非常宝贵的生化工具，因为它使一个目标GPCR的单密码子扫描。我们特别关注这里的UAA，AZF，其中有一个反应阿紫的多功能性做的部分。除了 ​​作为一个独特的红外（IR）探测器，8,9 AZF也可以作为一个可光活化交联剂通过与邻近的伯胺或脂族氢的反应。此外，生物惰性叠氮基可以参与作为生物正交标记反应选择性的化学处理。在这里，我们提出了举例说明位点特异性结合AZF的有用的应用程序转换为G蛋白偶联受体，例如有针对性的光交诱捕受体 - 配体复合物，并通过G蛋白偶联受体抗原生物正交标记和荧光标记的策略进行修改。 光活化试剂已被用于研究生物系统自1960年以来，10在这期间，受体-配体的交联实验丰盈已报道研究G蛋白偶联受体复合物，其中大部分涉及利用光亲和配体。11,12然而，这些应用技术上的限制，因为它们必需参数E的合成配体带有交联基团的13-15再者，在配位体的交联基团，它是具有挑战性的，以确定在G蛋白偶联受体的交联点的位置。位点特异性地引入光交联基团如UAAs到使用琥珀密码子抑制技术的蛋白质是一种有价值的进步。16,17我们开发了一种光致交联的技术，以确定对涉及的受体-配体复合物的形成是一个受体的结合界面通过引入光不稳定基团引入GPCR的活细胞。18,19在这里，我们描述的实验方案和数据分析的方法，应用该目标光交联技术，以确定一个小分子配体的结合位点，氚标记马拉韦罗，对CCR5。该方法利用了对配体的放射性的手柄，除了保留配体的天然化学结构的精确定量。 基于荧光技术直接探测受体的构象状态支持受体激活的结构基础的精确理解。20,21然而，拥有技术引进荧光标记成G蛋白偶联受体的灵活性，位点特异性是有限的。我们感兴趣的是使用生物正交化学修饰策略，以促进G蛋白偶联受体信号复合物的单分子检测（SMD），22叠氮基可以参与生物正交化学物质，如施陶丁格-Bertozzi教授结扎，23，24无铜应变推动叠氮炔环加成反应（SpAAC）25和铜催化的叠氮-炔环加成反应（CuAAC）26在这里，我们重点放在涉及叠氮化物和磷化氢之间的特异性反应的施陶丁格-Bertozzi教授连接反应。我们展示了使用两种不同的膦衍生物，缀合的肽表位（FLAG肽）或荧光标记的（荧光素），以实现GPCR的位点特异性修饰。 我们以前优化的视紫红质AZF变种利用X-射线晶体结构和动态模拟来选择是溶剂暴露目标站点的站点特定标记的条件。27，28，我们也说明了实现无背景标签的可行性，施陶丁格- Bertozzi教授结扎29我们在这里展示用于实现该被固定在免疫亲和基质，并随后通过在凝胶荧光可视化的洗涤剂增溶的受体荧光标记的一般方法。此外，我们证明这个标签战略，以确定适合于对未知结构，CCR5的受体标记位置的有用的扩展。这是通过使用一个依赖于的AZF-GPCR在基于细胞的半高通量形式的变体生物正交变型的靶向肽的表位标记的战略。30这方法利用细胞表面的ELISA的多步骤的检测性能监视标记的事件。 我们在这里讨论的方法是通用的，原则上可以适用于使用琥珀密码子抑制技术与AZF注册成立的任何G蛋白偶联受体。在这里给出的细节，我们涉及使用非天然氨基酸的诱变方法和其后续的应用，以促进G蛋白偶联受体的结构和动力学研究UAAs如AZF结合受体的哺乳动物细胞表达的步骤的协议。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmid pSVB. Yam</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2008)</td>
		</tr>
		<tr>
			<td>Plasmid pcDNA.RS for azF</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2009)</td>
		</tr>
		<tr>
			<td>Plasmids pMT4.Rho and pcDNA 3.1.CCR5</td>
			<td></td>
			<td></td>
			<td>Optionally contain the amber stop codon (TAG) at a desired position</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td></td>
			<td></td>
			<td>Adherent cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco</td>
			<td>10566</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine Plus</td>
			<td>Invitrogen</td>
			<td>Lipofectamine: 18324-012 
			Plus: 11514-015</td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine</td>
			<td>Chem-Impex International</td>
			<td>6162</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Site-specific genetic incorporation of unnatural amino acids into GPCRs materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Maxima ML-3500S UV-A lamp</td>
			<td>Spectronics Corporation</td>
			<td></td>
			<td>azF is activated by 365-nm light</td>
		</tr>
		<tr>
			<td>Hank&#39;s Buffered Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Irvine Scientific</td>
			<td>9319</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Roche</td>
			<td>3117405001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tritium-labeled ligand</td>
			<td></td>
			<td></td>
			<td>From collaborator (Grunbeck et al., 2012)</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>1% (w/v) sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Trans-Blot SD apparatus</td>
			<td>Biorad</td>
			<td></td>
			<td>Apparatus for semi-dry transfer</td>
		</tr>
		<tr>
			<td>Immobilon polyvinylidene difluoride (PVDF) membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat powered milk</td>
			<td>Fisher Scientific</td>
			<td>NC9934262</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Aldrich</td>
			<td>274348</td>
			<td></td>
		</tr>
		<tr>
			<td>Ecoscint A</td>
			<td>National Diagnostics</td>
			<td>LS-273</td>
			<td>Scintillation fluid</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>333726</td>
			<td></td>
		</tr>
		<tr>
			<td>LKB Wallac 1209 Rackbeta Liquid Scintillation Counter</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Beta-Scintillation counter</td>
		</tr>
		<tr>
			<td>Anti-rhodopsin 1D4 mAb</td>
			<td>National Cell Culture Center</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated anti-mouse IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyblot CL AR film</td>
			<td>Denville</td>
			<td>E3018</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Targeted photocrosslinking materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Invitrogen</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr>
			<td>FLAG-triarylphosphine</td>
			<td>Sigma</td>
			<td>GPHOS1</td>
			<td></td>
		</tr>
		<tr>
			<td>M2 FLAG mAb</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CCR5 2D7 mAb</td>
			<td>BD Biosciences</td>
			<td>555990</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Costar</td>
			<td>3601</td>
			<td>Clear bottom, high binding EIA/RIA</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Calcium, Magnesium)</td>
			<td>Gibco</td>
			<td>14040</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>EMS</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red</td>
			<td>Invitrogen</td>
			<td>A12222</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>DE Healthcare Products</td>
			<td>97-93399</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFluor II fluorescence multi-well plate reader</td>
			<td>Perseptive Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Targeted peptide-epitope tagging and cell surface ELISA materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Fluorescein-phosphine</td>
			<td></td>
			<td></td>
			<td>(Huber et al., submitted 2012)</td>
		</tr>
		<tr>
			<td>Nonapeptide (C9 peptide)</td>
			<td>AnaSpec</td>
			<td>62190</td>
			<td>Peptide mimicking the 1D4-epitope NH2-TETSQVAPA-COOH</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Confocal Typhoon 9400 fluorescence scanner</td>
			<td>GE Healthcare</td>
			<td>discontinued</td>
			<td>Scanner with 488-nm wavelength laser</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Fluorescent labeling materials</td>
		</tr>
	</tbody>
</table>

genetically-encoded molecular probes to study g protein-coupled receptors

以促进G蛋白偶联受体（GPCR）信号复合物的结构和动力学研究，新的方法需要引入翔实的探针或标记入表达的受体不扰乱受体的功能。我们使用琥珀密码子抑制技术，基因编码的非天然氨基酸， 对 -叠氮基-L-苯丙氨酸（AZF）在异源表达在哺乳动物细胞中的GPCR各种目标的位置。叠氮基的通用性是这里示出在不同的应用程序来研究G蛋白偶联受体在其天然细胞环境或洗涤剂溶解的条件下进行。首先，我们展示了基于细胞的靶向光交联技术，以确定在G蛋白偶联受体，其中一个氚标记的小分子配体交联到一种基因编码的叠氮基的氨基酸的配体结合口袋的残基。然后，我们通过生物正交的Staudinger-Bertozzi教授结扎反应表明G蛋白偶联受体的位点特异性修饰化用膦衍生物为目标的叠氮基。我们将讨论用于使用全细胞系的ELISA方法在培养及其检测表达的膜蛋白的靶向肽的表位标记的一般策略。最后，我们证明了AZF-G蛋白偶联受体可以选择性标记的荧光探针。讨论的方法是通用的，因为它们可以在原则上被应用到任何的任何氨基酸位置表示的GPCR来询问活性信号传导复合物。

我们所用的非天然氨基酸的诱变方法，以位点特异性地引入分子探针进入的GPCR使用琥珀密码子抑制技术。 图1中的方案概述的显着的步骤的方法，并结合了多功能UAA， 对-叠氮基-L-苯丙氨酸（AZF），进GPCRs的各种应用。的AZF-G蛋白偶联受体在哺乳动物细胞中的表达可让有针对性的光交联配体，并通过生物 ​​正交标记化学针对性的肽表位标记或G蛋白偶联受体的荧光修改。...

Watch this Scientific Journal Video about Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors at JoVE.com

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

在GPCR的各种目标的位置，我们基因编码的非天然氨基酸，对 - 叠氮基-L-苯丙氨酸，并显示在叠氮基团中的不同的应用程序的通用性。这些包括有针对性的光交联技术，以确定在一个G蛋白偶联受体的配体结合口袋的残基，和位点特异性生物正交的GPCR与肽表位标签或荧光探针的修饰。

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

genetically-encoded-molecular-probes-to-study-g-protein-coupled

Research

JoVE Journal

Biology

15.1K 次观看.  The Rockefeller University. 在GPCR的各种目标的位置，我们基因编码的非天然氨基酸，对 - 叠氮基-L-苯丙氨酸，并显示在叠氮基团中的不同的应用程序的通用性。这些包括有针对性的光交联技术，以确定在一个G蛋白偶联受体的配体结合口袋的残基，和位点特异性生物正交的GPCR与肽表位标签或荧光探针的修饰。 

视频: Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

量化在G蛋白偶联受体激动剂活性

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

水培：一个多功能的系统来研究营养分配和植物响应养分有效性及暴露于有毒元素

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...