Name: gramicidin-based fluorescence assay; for determining small molecules potential for modifying lipid bilayer properties
Uploaded: 2010-10-13T10:00:00
Description: Watch this Scientific Journal Video about Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties at JoVE.com

我们感谢许多刺激讨论迈克尔J.布鲁诺，拯救Rusinova和Jon T.袋。从美国国立卫生研究院，R01GM021342和阿塞拜疆恢复和重建补充R01GM021342 - 35S1，约西亚梅西，小基金会OSA的财政支持;三我的HII中巴方案;以及鸢尾属莱弗里特南伍德沃思医学科学家奖学金和美国国立卫生研究院的MSTP授予GM07739为RK。

1。生成蝼蚁充满脂质体<ol><li>第1天，除去有机溶剂从血脂。 </li><li>从冰箱中取出血脂和让它平衡至室温。 </li><li>加入0.6毫升25毫克/毫升（1,2 dierucoyl - SN -甘油- 3 - phosphocholine）在氯仿溶液25 ml圆底烧瓶中的脂质。 </li><li>不断旋转烧瓶，而干燥氮气保护下，直到所有的氯仿蒸发，薄薄的白色膜脂质大衣整个降低一半烧瓶。 </li><li>干燥在真空状态下dessicator一夜之间。 </li></ol><ol><li>第2天，准备将?...

<ol>
	<li>Andersen, O. S., Giebisch, G. H., Purcel, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+transport+through+simple+membranes.">Ion transport through simple membranes.</a> Renal Function. , (1978).</li><li>Andersen, O. S., Koeppe, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayer+thickness+and+membrane+protein+function:+An+energetic+perspective.">Bilayer thickness and membrane protein function: An energetic perspective.</a> Annu. Rev. Biophys. Biomol. Struct. 36, 107-130 (2007).</li><li>Andersen, O. S., Koeppe, R. E., Roux, B., Chung, S. -. H., Andersen, O. S., Krishnamurthy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gramicidin+channels.+Versatile+tools.">Gramicidin channels. Versatile tools.</a> Biological Membrane Ion Channels: Dynamics, Structure, and Applications. , (2007).</li><li>Berberan-Santos, M. N., Bodunov, E. N., Valeur, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+functions+for+the+analysis+of+luminescence+decays+with+underlying+distributions+1.+Kohlrausch+decay+function+(stretched+exponential.">Mathematical functions for the analysis of luminescence decays with underlying distributions 1. Kohlrausch decay function (stretched exponential.</a> Chem. Phys. 315, 171-182 (2005).</li><li>Bruggemann, E. P., Kayalar, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+molecularity+of+the+colicin+E1+channel+by+stopped-flow+ion+flux+kinetics.">Determination of the molecularity of the colicin E1 channel by stopped-flow ion flux kinetics.</a> Proc. Natl. Acad. Sci. USA. 83, 4273-4276 (1986).</li><li>Bruno, M. J., Koeppe, R. E., Andersen, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Docosahexaenoic+acid+alters+bilayer+elastic+properties.">Docosahexaenoic acid alters bilayer elastic properties.</a> Proc. Natl. Acad. Sci. USA. 104, 9638-9643 (2007).</li><li>Buboltz, J. T., Feigenson, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+for+the+preparation+of+liposomes:+rapid+solvent+exchange.">A novel strategy for the preparation of liposomes: rapid solvent exchange.</a> Biochim. Biophys. Acta. 1417, 232-245 (1999).</li><li>Gutknecht, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadmium+&#38;+thallous+ion+permeabilities+through+lipid+bilayer+membranes.">Cadmium &#38; thallous ion permeabilities through lipid bilayer membranes.</a> Biochim. Biophys. Acta. 735, 185-188 (1983).</li><li>Ing&#243;lfsson, H. I., Koeppe, R. E., Andersen, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+is+a+modulator+of+bilayer+material+properties.">Curcumin is a modulator of bilayer material properties.</a> Biochemistry. 46, 10384-10391 (2007).</li><li>Keser&#252;, G. M., Makara, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+lead+discovery+strategies+on+the+properties+of+drug+candidates.">The influence of lead discovery strategies on the properties of drug candidates.</a> Nat. Rev. Drug Discov. 8, 203-212 (2009).</li><li>Leeson, P. D., Springthorpe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+drug-like+concepts+on+decision-making+in+medicinal+chemistry.">The influence of drug-like concepts on decision-making in medicinal chemistry.</a> Nat. Rev. Drug Discov. 6, 881-890 (2007).</li><li>Lundb k, J. A., Collingwood, S. A., Ing&#243;lfsson, H. I., Kapoor, R., Andersen, O. S., S, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+bilayer+regulation+of+membrane+protein+function:+gramicidin+channels+as+molecular+force+probes.">Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes.</a> J. R. Soc. Interface. 7, 373-395 (2010).</li><li>Moore, H. P. &. a. m. p. ;. a. m. p., Raftery, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+spectroscopic+studies+of+cation+translocation+by+Torpedo+acetylcholine+receptor+on+a+time+scale+of+physiological+relevance.">Direct spectroscopic studies of cation translocation by Torpedo acetylcholine receptor on a time scale of physiological relevance.</a> Proc. Natl. Acad. Sci. USA. 77, 4509-4513 (1980).</li><li>Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+specificity+of+the+gramicidin+channel+and+the+thallous+ion.">Ionic specificity of the gramicidin channel and the thallous ion.</a> Biochim. Biophys. Acta. 401, 540-544 (1975).</li><li>O'Connell, A. M., Koeppe, R. E., Andersen, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+gramicidin+channel+formation+in+lipid+bilayers:+transmembrane+monomer+association.">Kinetics of gramicidin channel formation in lipid bilayers: transmembrane monomer association.</a> Science. 250, 1256-1259 (1990).</li><li>S&#248;gaard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAA+receptor+function+is+regulated+by+lipid+bilayer+elasticity.">GABAA receptor function is regulated by lipid bilayer elasticity.</a> Biochemistry. 45, 13118-13129 (2006).</li><li>Waring, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+optimum+lipophilicity+and+molecular+weight+ranges+for+drug+candidates-Molecular+weight+dependent+lower+logD+limits+based+on+permeability.">Defining optimum lipophilicity and molecular weight ranges for drug candidates-Molecular weight dependent lower logD limits based on permeability.</a> Bioorg. Med. Chem. Lett. 19, 2844-2851 (2009).</li></ol>

我们已经确定的双层修改药品和其他小双亲的潜力表现出快速的荧光为基础的检测。修改双层属性的化合物有可能间接的，非特异性的方式改变膜蛋白的功能，可能有助于“脱靶”的药物作用。该实验利用双层跨越蛋白质检测的12个双层属性的变化进行探测的短杆菌肽渠道的力量。使用基于荧光检测得到的结果与单通道12 GA实验结果吻合良好，表明该方法可用于筛选化合物库以及?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>ANTS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-350</td>
<td>&nbsp;</td>
<tr>
<td>gramicidin</td>
<td>&nbsp;</td>
<td>Sigma Chemical Co</td>
<td>G-5002</td>
<td>&nbsp;</td>
<tr>
<td>1,2-dierucoyl-sn-glycero-3-phosphocholine</td>
<td>&nbsp;</td>
<td>Avanti Polar Lipids</td>
<td>850398C</td>
<td>&nbsp;</td>
<tr>
<td>Mini-Extruder kit</td>
<td>&nbsp;</td>
<td>Avanti Polar Lipids</td>
<td>610000</td>
<td>&nbsp;</td>
<tr>
<td>PD-10 Desalting column</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich Made by GE Healthcare</td>
<td>54805</td>
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gramicidin-based fluorescence assay; for determining small molecules potential for modifying lipid bilayer properties

用来调节生物功能的许多药物和其他小分子吸附在双层/溶液界面，从而改变脂质双层属性的双亲。这是很重要的，因为膜蛋白大力加上他们的主机双层的疏水相互作用。双层属性的变化，从而改变膜蛋白的功能，它提供了一种间接的方式为双亲调节蛋白质的功能，并为“脱靶”的药物作用的可能机制。之前我们已经开发出一种检测脂双层属性的变化，使用线性探头3,12短杆菌肽通道的电生理检测。短杆菌肽渠道transbilayer二聚体的两个非进行亚基组成的小型蛋白质。他们是敏感，在他们的膜环境，这使得他们在脂质双层属性的监测变化的强大探头双层跨越蛋白质检测变化。我们现在表现出的检测双层属性的变化，使用相同的渠道为探针的荧光分析法。该试剂盒是基于测量荧光猝灭的时间，当然，由于通过短杆菌肽通道进入一个淬灭的荧光装的大unilamellar水泡。我们使用荧光指示灯/淬灭对8 -萘胺-2,3,6 - trisulfonate（蚂蚁）/ TL +已成功地用于其他荧光猝灭测定 5,13 。 TL +渗透脂质双层慢慢8，但很容易通过开展短杆菌肽通道 1,14 。该方法可扩展性和适宜双层扰动，和潜在的“目标”，效果的小分子机理研究和高通量筛选。我们发现，使用这种方法的结果与前 12电结果吻合良好。

Watch this Scientific Journal Video about Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties at JoVE.com

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

我们介绍一个快速荧光检测监控率作为衡量短杆菌肽通道活性的荧光猝灭。短杆菌肽通道，作为分子的力传感器监测双层跨越蛋白质检测中的脂质双层属性的变化。

短杆菌肽为基础的荧光分析法，确定小分子的修改脂质双层属性的潜在

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

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