作者想感谢埃莉诺弗莱明和杰西卡陈有益的讨论。经费是由国家过敏和传染病研究所（NIH - NIAID的）奖R15AI079685和研究公司科特雷尔科学学院奖。由霍华德休斯医学研究所和斯特利基金，附加学生提供了支持。

1。制备大Unilamellar脂质囊泡（LUVs） <ol><li>准备LUVs作为细胞膜模拟检测19。 </li><li>混合磷脂（POPC，760.10克/摩尔），磷脂（POPG. 770.99克/摩尔），5 dimethylaminonaphthalene - 1 -磺酰磷脂（DNS教皇，994.350克/摩尔）（阿凡提极性脂）在氯仿中溶解在50 ：45:5的比例。由于每个检测需要单独编制的实验和控制LUVs，两个脂质蛋糕小瓶应准备。 0.376毫克总脂质（POPC 0.188毫克，0.169毫克POPG，和0.019毫克DNS教皇）通常是足够的，以弥补单一的易位试验足够的囊泡。 </li><li>去氯仿使用的N 2气流蒸发。 </li><li> 9-14小时在真空干燥器干燥脂质蛋糕。 </li><li>准备一只股票10毫米HEPES缓冲溶液（10 mM的HEPES，238.3克/摩尔，45毫米氯化钠，58.44克/摩尔，1 mM EDTA的372.24克/摩尔pH值7.4）。 HEPES缓冲是ST有色在4 ° C。 </li><li>准备在10毫米准备HEPES缓冲液原液的0.4毫米猪胰蛋白酶（23.3公斤/摩尔）。胰蛋白酶是储存在-20 ° C，并在实践中，通常不接受使用前超过2冻融。 </li><li>准备在10 mM的HEPES缓冲的解决方案和存储于-20 ° C原液的4.0毫米鲍曼- Birk胰蛋白酶抑制剂（8公斤/ MOL）在我们的使用，这种股票是每月补充和通常不接受前再次它是由冻结/解冻周期超过3。 </li><li>重组实验multilamellar囊泡（MLV更具）加入等体积的0.4毫米胰蛋白酶的股票和10毫米的HEPES缓冲溶液干脂质蛋糕的胰蛋白酶溶液在0.2毫米。对于单个样品的0.376毫克总脂，150μL胰蛋白酶溶液和150μLHEPES缓冲的混合物就足够了。 </li><li>在加入同等体积为0的解决方案，0.2毫米胰蛋白酶和2.0毫米鲍曼- Birk胰蛋白酶抑制剂的重组的控制MLV更具。4毫米的胰蛋白酶股票和4.0毫米的胰蛋白酶抑制剂的干脂质蛋糕。对于单个样品的0.376毫克总脂，150μL胰蛋白酶溶液和150μL胰蛋白酶抑制剂的混合物就足够了。 </li><li> MLV的传输解决方案，从玻璃瓶到1.7 ml离心管。 </li><li>主题MLV 5冻结/解冻周期的解决方案，在液氮中。 </li><li>准备放置两个过滤器支持在每个聚四氟乙烯挤出一块蘸HEPES缓冲脂挤出机（阿凡提极性脂）。放置一个0.1微米的毛孔（滤纸，英国），两者之间的聚四氟乙烯挤出件在nuclepore跟踪蚀刻膜，并将其放在金属挤出机筒挤出机内件。紧缩的挤出机筒的金属的顶端持有人，并把它放在前两个挤出件甜甜圈间隔。 </li><li>填写挤出机内的空隙体积500μL，10毫米的HEPES使用250μL玻璃注射器，以防止损失囊泡样品的缓冲区。&lt;/ LI&gt; <li>每个MLV的样品装入挤出机，并推动通过膜的样品，至少有21次，以确保统一的unilamellar囊泡大小。 LUVs获得MLV解决方案以下挤出。 </li><li>储存于4 LUVs ° C和48小时内使用。 </li></ol> 2。量化LUV浓度<ol><li> LUV浓度进行了量化，确定样品中的 20的总磷含量。 </li><li>准备8.9 NH 2 SO 4 nanopure水，在室温下存储。 </li><li>准备了2.5％W / v钼酸铵六（588.04克/摩尔）nanopure水的解决方案。准备了10％W / V抗坏血酸（176.1克/摩尔）在nanopure水的解决方案。从覆盖的管带箔的光线照射保护的抗坏血酸溶液。长达2个月，保存于4 ° C的两种解决方案。 </li><li>准备为0.65毫米的磷酸盐缓冲溶液（二氢无水磷酸钠，120克/摩尔）。 </li><li>准备6个标准TU。BES。标准应包含0.0μL，50μL（0.0325μmoles），100μL（0.065μmoles），175μL（0.114μmoles），250μL（0.163μmoles），或350μL磷溶液（0.228μmoles）。 </li><li>准备LUV一式三份样品管。每个样品管应包含约0.1 LUVsμmoles。实验组和对照我必须单独计量的浓度。 </li><li>要纠正任何Bowman - Birk抑制剂粉干磷盐，控制LUV必须准备的空白。空白应该包含相同体积的0.2毫米trypsin/2.0 mM的Bowman - Birk抑制剂的解决方案，如被放置在控制LUV样品管。 </li><li>将450微升8.9 NH 2 SO 4到每个样品，标准和空白管。 </li><li>热样品，标准，和25分钟的空白，在175-210 ° C在烤箱。 </li><li>删除所有的管子，让他们冷却。加入150μL30％H 2 O 2涂BES。 </li><li>热样品，标准和30分钟的空白，在175-210 ° C。 </li><li>取出的样品，标准，并从热的空白。如果任何解决方案都尚未无色，添加50μL的H 2 O 2所有热管和一个额外的15分钟在175-210 ° C。 </li><li>添加3.9毫升nanopure H 2 O，0.5毫升钼酸铵溶液和0.5 mL的抗坏血酸溶液每管。 </li><li>在沸水浴中加热5-7分钟管。 </li><li>测量每个标准和样品使用Agilent 8453紫外可见分光光度计在820 nm的吸光度。管含有0.0 mmoles磷应使用所有标准和实验的空白LUV样本。管含有0.2毫米trypsin/2.0 mM的Bowman - Birk抑制剂的解决方案应作为控制的空白LUV样本。 </li><li>线性吸光度与磷含量的标准曲线，可以生产和使用，计算出总的磷酸化orus内容和LUV样品的浓度。 </li></ol> 3。准备肽解决方案<ol><li>在nanopure H 2 O的溶解肽在本实验中使用的描述肽必须包含一个色氨酸残基。 </li><li>测量肽的解决方案一式三份于282 nm的吸光度。色氨酸5700 M -1 cm -1处的摩尔消光系数计算肽浓度。 </li><li> nanopure水稀释到终浓度为30微米的肽。贮存于-20 ° C </li></ol> 4。易位量化<ol><li>准备在1.7 ml离心管实验组和对照样本。每个解决方案中添加足够的LUVs终浓度为250微米。加入足够的Bowman - Birk抑制剂的解决方案，使抑制剂终浓度为10倍大于胰蛋白酶浓度。添加足够的10毫米HEPES缓冲带来的最终解决方案的体积为450μL。 </li> &lt;李&gt;广场50μL成Starna微石英荧光细胞肽的解决方案。 LUVs和多肽之间的比例是高到足以确保几乎所有完整的肽是在足够接近接近到丹磺酰组给予的FRET信号，即使没有进入囊泡易位。这是类似的FRET在控制条件下观察到的肽和不转运入囊泡。 </li><li>新增实验或对照样品在试管的肽解决方案。这应该带来最终的肽浓度至3微米。 </li><li>开始25分钟的荧光动力学实验或控制后除了实验立即LUV解决方案，至少一次到每秒荧光阅读。激发波长在280 nm，发射波长为525 nm，温度设定为25 ° C使用Cary Eclipse荧光分光光度计，我们设置的光电倍增管的灵敏度高。 </li></ol><html> 5。生成定量易位比例<ol><li>定义为初始荧光（F O）后15秒的数据收集荧光读。在我们的仪器设置中，我们发现，第一个15秒采集的荧光数据是不可靠的，由于混合，关闭样品室，和其他干扰后，样品除了系统。 F O除以每个随后的阅读，以获得在每个时间点相对荧光（F / F 0 ）。 </li><li>平均所有实验的最后一分钟的相对荧光读数获得最终的平均相对荧光<img alt="[F / FO]平均" src="/files/ftp_upload/3571/3571eq1.jpg" /> 。 </li><li>除以<img alt="[F / FO]平均" src="/files/ftp_upload/3571/3571eq1.jpg" />由控制样品<img alt="[F / FO]平均" src="/files/ftp_upload/3571/3571eq1.jpg" />实验样品获得一个正确的最终荧光值。 </li></ol>乐“&gt; 6。代表性的结果图2显示了这个有代表性的肽，​​表现出强劲的易位的检测结果。在这个实验中的信号（黑色线），显示了在经过一段时间的FRET信号的显着下降。但是，重要的是要控制的FRET信号，由于不完整的胰蛋白酶抑制或易位能力无关的其他因素的潜在损失。为此，我们也总是测量FRET信号之间的肽和LUVs含胰蛋白酶和鲍曼- Birk胰蛋白酶抑制剂（灰色轨迹，图2）。在我们的手中，它已被控制每一个实验运行时执行每肽。这使我们能够清楚正确的信号的任何变化，由于脂质囊泡制剂或仪器噪声，可用于相对较弱的荧光信号通常是在观察这些浓度显著之间的任何分歧。 控制EXPE的重要性riment使用LUVs含有胰蛋白酶抑制剂是强调了在图3所示的数据。在这种情况下，肽信号减少类似数额，在图2实验样品（黑色曲线）。然而，控制的示例显示了这种肽更快速的减少，因此其净易位是较低的。 我们的协议第5条描述了一个简单的方法，从这个实验中量化易位。更高的易位比率肽转运有效的指标，图2所示为细胞穿透肽的易位的比例是1.16，而弱转位肽易位比率接近1。根据我们的经验，不同的囊泡准备进行3次独立实验的标准误差为0.01至0.06的范围内。 <img alt="图1" src="/files/ftp_upload/3571/3571fig1.jpg" /> 图1。示意图translocat离子检测实验LUV样本（ 一 ）掺杂荧光丹磺酰教皇（黑网吧），包含封装胰蛋白酶（剪刀）。胰蛋白酶抑制剂（红色圆圈）是用来抑制胰蛋白酶外LUVs。 LUVs暴露肽（紫色）。肽与LUV膜产生的FRET信号（绿色），跌幅translocting肽遇到不羁的内部胰蛋白酶。胰蛋白酶和胰蛋白酶抑制剂是控制封装LUV样品（B）来衡量的FRET信号，以易位无关的跌幅。 <img alt="图2" src="/files/ftp_upload/3571/3571fig2.jpg" /> 图2。一个细胞穿透肽的有代表性的数据 。一个代表转位的抗菌pepide，显示了FRET的信号相比，其控制的显著下降。 <img alt="图3" src="/files/ftp_upload/3571/3571fig3.jpg" /> 图3。突出控制的重要性。代表性的数据不完全抑制非转位肽胰蛋白酶消化，导致在FRET的信号随着时间的推移下降，在实验组和对照LUV的解决方案。由于控制和实验的痕迹之间的差异相对较小，这种突变的特点是作为一个弱转位肽。

<ol>
	<li>Henriques, S. T., Melo, M. N., Castanho, 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=Cell-penetrating+peptides+and+antimicrobial+peptides:+how+different+are+they?.">Cell-penetrating peptides and antimicrobial peptides: how different are they?.</a> Biochem. J. 399 (1), 1-1 (2006).</li><li>Trehin, R., Merkle, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chances+and+pitfalls+of+cell+penetrating+peptides+for+cellular+drug+delivery.">Chances and pitfalls of cell penetrating peptides for cellular drug delivery.</a> Eur. J. Pharm. Biopharm. 58 (2), 209-209 (2004).</li><li>Temsamani, J., Vidal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+cell-penetrating+peptides+for+drug+delivery.">The use of cell-penetrating peptides for drug delivery.</a> Drug Discov. Today. 9 (23), 1012-1012 (2004).</li><li>Hale, J. D., Hancock, 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=Alternative+mechanisms+of+action+of+cationic+antimicrobial+peptides+on+bacteria.">Alternative mechanisms of action of cationic antimicrobial peptides on bacteria.</a> Expert Review of Anti-Infective Therapy. 5 (6), 951-951 (2007).</li><li>Nicolas, P., Rosenstein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+host+defense+peptides.">Multifunctional host defense peptides.</a> FEBS J. 276 (22), 6464-6464 (2009).</li><li>Boman, H. G., Agerberth, B., Boman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Action+on+Escherichia-Coli+of+Cecropin-P1+and+Pr-39,+2+Antibacterial+Peptides+from+Pig+Intestine.">Mechanisms of Action on Escherichia-Coli of Cecropin-P1 and Pr-39, 2 Antibacterial Peptides from Pig Intestine.</a> Infection and Immunity. 61 (7), 2978-2978 (1993).</li><li>Park, C. B., Kim, H. S., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+action+of+the+antimicrobial+peptide+buforin+II:+Buforin+II+kills+microorganisms+by+penetrating+the+cell+membrane+and+inhibiting+cellular+functions.">Mechanism of action of the antimicrobial peptide buforin II: Buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions.</a> Biochemical and Biophysical Research Communications. 244 (1), 253-253 (1998).</li><li>Bobone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thin+line+between+cell-penetrating+and+antimicrobial+peptides:+the+case+of+Pep-1+and+Pep-1-K.">The thin line between cell-penetrating and antimicrobial peptides: the case of Pep-1 and Pep-1-K.</a> J. Pept. Sci. 17 (5), 335-335 (2011).</li><li>Marks, J. R., Placone, J., Hristova, K., Wimley, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+Membrane-Translocating+Peptides+by+Orthogonal+High-throughput+Screening.">Spontaneous Membrane-Translocating Peptides by Orthogonal High-throughput Screening.</a> J. Am. Chem. Soc.. , (2011).</li><li>Rosenbluh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+histone+proteins+across+lipid+bilayers+and+Mycoplasma+membranes.">Translocation of histone proteins across lipid bilayers and Mycoplasma membranes.</a> J. Mol. Biol. 345 (2), 387-387 (2005).</li><li>Bárány-Wallje, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+reassessment+of+penetratin+translocation+across+lipid+membranes.">A critical reassessment of penetratin translocation across lipid membranes.</a> Biophys. J. 89 (4), 2513-2513 (2005).</li><li>Henriques, S. T., Costa, J., Castanho, 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=Translocation+of+beta-galactosidase+mediated+by+the+cell-penetrating+peptide+pep-1+into+lipid+vesicles+and+human+HeLa+cells+is+driven+by+membrane+electrostatic+potential.">Translocation of beta-galactosidase mediated by the cell-penetrating peptide pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential.</a> Biochemistry. 44 (30), 10189-10189 (2005).</li><li>Orioni, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+perturbation+by+the+antimicrobial+peptide+PMAP-23:+a+fluorescence+and+molecular+dynamics+study.">Membrane perturbation by the antimicrobial peptide PMAP-23: a fluorescence and molecular dynamics study.</a> Biochim. Biophys. Acta. 1788 (7), 1523-1523 (2009).</li><li>Ladokhin, A. S., Jayasinghe, S., White, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+and+analyze+tryptophan+fluorescence+in+membranes+properly,+and+why+bother?.">How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?.</a> Anal. Biochem. 285 (2), 235-235 (2000).</li><li>Epand, R. M., Epand, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+as+models+for+antimicrobial+peptides.">Liposomes as models for antimicrobial peptides.</a> Methods Enzymol. 372, 124-124 (2003).</li><li>Kobayashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+novel+antimicrobial+peptide+buforin+2+with+lipid+bilayers:+Proline+as+a+translocation+promoting+factor.">Interactions of the novel antimicrobial peptide buforin 2 with lipid bilayers: Proline as a translocation promoting factor.</a> Biochemistry. 39 (29), 8648-8648 (2000).</li><li>Matsuzaki, K., Murase, O., Fujii, N., Miyajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+a+channel-forming+antimicrobial+peptide,+magainin+2,+across+lipid+bilayers+by+forming+a+pore.">Translocation of a channel-forming antimicrobial peptide, magainin 2, across lipid bilayers by forming a pore.</a> Biochemistry. 34 (19), 6521-6521 (1995).</li><li>Henriques, S. T., Costa, J., Castanho, 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=Re-evaluating+the+role+of+strongly+charged+sequences+in+amphipathic+cell-penetrating+peptides:+a+fluorescence+study+using+Pep-1.">Re-evaluating the role of strongly charged sequences in amphipathic cell-penetrating peptides: a fluorescence study using Pep-1.</a> FEBS Lett. 579 (20), 4498-4498 (2005).</li><li>Torchilin, V. P., Weissig, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Liposomes. , (2003).</li><li>Almeida, P. F., Pokorny, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avanti+Polar+Lipids,+Determination+of+Total+Phosphorus.">Avanti Polar Lipids, Determination of Total Phosphorus.</a> Biochemistry. 1686 (34), 8083-8083 (2009).</li><li>Kobayashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+translocation+mechanism+of+the+antimicrobial+peptide+buforin+2.">Membrane translocation mechanism of the antimicrobial peptide buforin 2.</a> Biochemistry. 43 (49), 15610-15610 (2004).</li></ol>

没有利益冲突的声明。

这里介绍的协议，可用于评估脂质囊泡的内部和外部的肽浓度的相对变化。这些变化与易位的能力。这个协议可以被用来确定细胞穿透肽作为药物载体的潜力。由于细胞穿透肽的兴趣的增长，这将是有趣的，看看如何采用定量的方式开发和使用方法，直接测量易位事件。 在我们的实验室中，我们发现，可以通过仔细监测几个特定方面的实验提高检测的一致性。首先，实验组?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 16:0-18:1 PG </td><td>阿凡提极性脂</td><td> 840457C </td></tr><tr><td> 1:18丹磺酰PE </td><td>阿凡提极性脂</td><td> 810330C </td></tr><tr><td> 16:0-18:1的PC </td><td>阿凡提极性脂</td><td> 850457C </td></tr><tr><td>猪胰蛋白酶</td><td>西格玛</td><td> T - 0303 </td></tr><tr><td>鲍曼Birk胰蛋白酶/糜蛋白酶抑制剂</td><td>西格玛</td><td> T - 9777 </td></tr><tr><td>小型挤出机</td><td>阿凡提极性脂</td><td> 610000 </td></tr><tr><td>钼酸铵（段） </td><td>阿法埃莎</td><td> 10811 </td></tr><tr><td> L -抗坏血酸</td><td>西格玛</td><td> A1417 </td></tr><tr><td>过氧化氢，30％的解决方案</td><td> Mallinckrodt化学品</td><td> 5240-05 </td></tr><tr><td> HEPES </td><td>西格玛</td><td> H - 3375 </td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S - 9625 &lt;/ TD&gt; </tr><tr><td> EDTA </td><td>西格玛</td><td> E5134 </td></tr><tr><td>的NaH 2 PO 4 </td><td>西格玛</td><td> S - 0751 </td></tr></tbody></table>

measuring peptide translocation into large unilamellar vesicles

有一个多肽的积极关注，容易交叉没有细胞膜，细胞膜上的受体1协助。其中许多被称为细胞穿透肽，这是经常为他们的潜力作为药物载体 1-3注意到。此外，有越来越多的利益通过非4,5膜裂解机制，特别是那些跨而不引起细胞裂解细菌细胞膜并杀死细胞与细胞内过程的干扰 6,7抗菌肽。事实上，越来越多的作者指出，细胞穿透和抗菌肽1,8之间的关系。膜移位和肽的结构，其转运能力之间的关系过程中的一个坚定的认识需要有效的，重复性检测易位。一些团体提出的方法来衡量大unilamel易位LAR脂质囊泡（LUVs）9-13。 LUVs作为有用的模式细菌和真核生物的细胞膜和肽 14,15荧光研究中经常使用。在这里，我们描述了我们的第一考虑如抗菌肽magainin和buforin二16,17，松崎和同事开发的方法的应用程序。除了这种方法为我们的协议，我们也提出一个简单的数据分析方法，量化易位使用这种检测能力。这种易位检测的比别人的优点是，它有可能提供膜易位率的信息，不需要另外一个荧光标签，可以改变肽属性18，含色氨酸的肽。简单地说，易位到脂质囊泡的能力是衡量本地色氨酸残基和D之间的福斯特共振能量转移（FRET）的功能与外部相关的蛋白质时，LUV ansyl磷脂膜（图1）。细胞穿透肽裂解，因为他们遇到不羁的胰蛋白酶封装的LUVs，LUV膜和下降的FRET信号解离。在DROP FRET为转位肽观察到的信号是明显高于观察LUVs含有胰蛋白酶和胰蛋白酶抑制剂，或当不自发地穿过脂膜是一种肽暴露在含有胰蛋白酶LUVs相同的肽。这种荧光的变化提供了直接量化，随着时间的推移肽移位。

Watch this Scientific Journal Video about Measuring Peptide Translocation into Large Unilamellar Vesicles at JoVE.com

Measuring Peptide Translocation into Large Unilamellar Vesicles

该协议的细节肽易位到大量的unilamellar脂质囊泡的定量测量方法。这种方法还提供了关于膜易位率的信息，可用于识别肽，有效地和自发地跨越脂质双层。

测量肽转运到大Unilamellar囊泡

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

measuring-peptide-translocation-into-large-unilamellar-vesicles

Research

JoVE Journal

Biology

13.8K 次观看.  Wellesley College. 该协议的细节肽易位到大量的unilamellar脂质囊泡的定量测量方法。这种方法还提供了关于膜易位率的信息，可用于识别肽，有效地和自发地跨越脂质双层。

视频: Measuring Peptide Translocation into Large Unilamellar Vesicles

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

宏基因组库的大型屏幕

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

科研

生物学

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大型插入环境基因组文库生产

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

偏光易位荧光蛋白爪蟾外胚层响应Wnt信号

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

检查Bcl-2家族大单层脂质体的功能

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

从巨 Unilamellar 泡中提取膜纳米管

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...