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，准备将纳入到囊泡的荧光团的范例。 </li><li>在NANO 3 ​​100毫米，25毫米的蚂蚁，（娜盐）10毫米的HEPES补充水分。使用硝酸或氢氧化钠调节pH至7.0。每个蚁分子2的Na +，总共为150毫米的Na +]。添加1.671毫升的电解液，得到一个10毫米脂质的悬浮液。 </li><li>封口膜和涡悬挂彻底。 </li><li>防止光样品用铝箔，并让在室温下，年龄在一夜之间。 </li></ol><ol><li>第3天，使unilamellar大泡和从外囊泡中删除所有的荧光。 </li><li>超声为低功耗sonicator 1分钟。 </li><li>冻融样本5-6次，每次5分钟，干冰和温暖（〜50 ° C）水中5分钟。 </li><li>挤出的脂质悬挂使用阿凡提小型挤出机。设置一个0.1微米的聚碳酸酯过滤器和过滤器支持（见挤出机手册）的小型挤出机。挤出暂停来回21次暂停结束了相反的注射器。当多个批次的挤压，永远记住加载具有相同的注射器样本。多云初步脂质悬挂在挤出过程中，应该成为几乎半透明（它仍然是黄色由于蚁荧光基团）。如果挤压突然变得更容易，需要较少的压力将通过过滤器，过滤器可能已破裂，将需要更换。 </li><li>删除运行一个使用重力协议的PD - 10脱盐柱挤压悬挂外部蚁。 20-30毫升钠缓冲平衡列（NANO 3 ​​140毫米，10毫米的HEPES，pH值7.0，记得用硝酸或氢氧化钠调节pH）。挤压脂悬液加入1.5毫升的列，让它渗入。带来加入1毫升钠缓冲的总样本量2.5毫升。后缓冲区是完全纳入列，并没有泄漏出来，3毫升钠缓冲洗脱脂质体，并收集产生的样本。蚁充满LUV原液应包含有关血脂4-5毫米，并出现半透明的乳白色。保护样品的光带箔。 </li><li>如果股票的解决方案是不立即使用，储存在13 ° C为最多7天。警告：不要冻结或冷却低于其液体晶相的转变温度（〜12 ° C），因为这会破坏囊泡的完整性，并会泄漏出来的荧光脂质的解决方案。 </li></ol> 2。混合荧光解决方案<ol><li> 24小时前使用，稀释的脂质体和短杆菌肽孵育（13℃）。的短杆菌肽脂质囊泡的内层和外层膜之间的平衡是一个缓慢的过程，需要很长的潜伏期。 </li><li>彻底涡蚂蚁LUV充满原液作为LUVs密度&gt; 1克/毫升，因此将沉积物中的解决方案。 </li><li>混合蝼蚁充满LUV钠缓冲库存为1:20。在一天的实验使用的所有脂质体混合在一起在一个烧瓶中，以提高样品的均匀性。 </li><li>分离出的脂质体的3 / 4，并添加260 nm的短杆菌肽在DMSO预稀释（500微克/毫升）。 </li><li>余下的1 / 4的相同体积的DMSO（不含短杆菌肽）添加在所有样本中不断保持溶剂浓度。 </li></ol> 3。设立荧光仪器<ol><li>我们使用温度控制应用光物理SX.20停流荧光光谱仪测量荧光猝灭率。 </li><li>打开仪器提前一个小时，以便热身。的组件包括：计算机，电子控制单元，水浴（冷却器和加热器），灯电源和氮气罐。 </li><li>设置使用临数据SX软件的拍摄条件。 </li><li>设置1 / 1激发单色激发波长为352纳米，狭缝宽度光圈。 </li><li>使用λ= 455 nm的高通滤波器，以记录排放。 </li><li>使用“保持压力”的设置，使仪器保持在录制过程中的氮气压力，从而防止在几毫秒的压力振荡（和录音制品）。 </li><li>设置“钛我“，以1秒和”点“到5000。 </li><li>使用重复框和调整重复次数，我们通常使用9日和13重复，分别为缓冲区和淬灭运行。 </li><li>水浴温度应设置为25 ° C，温度应在SX软件的监控。 </li><li>设置驱动器的体积为120μL，这意味着仪器，每次运行一个示例，它混合了两个注射器60μL。有了这个工具的安装，120μL，卷给出了一个死区时间≈1.2毫秒。 </li><li>蚂蚁脂质体的输出到8点左右，调整荧光检测的高电压（增益）。对于一个标准的实验混合物的增益应400-420五，有些药物是荧光灯，如他们能饱和的荧光信号。在这种情况下，增益/电压应下降。 </li><li>始终加载到左边的注射器，并在合适的注射器缓冲/淬灭荧光样品。确保彻底涡含有脂质体装载前，所有样品，并意识到，随着时间的推移LUVs，将落户在注射器中。 </li></ol> 4。做一个实验<ol><li>准备一个蝼蚁加载LUVs与样本，无论是溶剂或化合物，并加载到一起要么钠缓冲或TL -淬灭（50毫米，94 毫米纳米 3 3 TlNO，在pH 7.0的10毫米的HEPES ）分光光度计。 </li><li>使用1.5 ml稀释蚂蚁LUV解决方案，或没有，准备在前一天的短杆菌肽。 </li><li>添加所需浓度的化合物或溶剂的控制。保持溶剂浓度到最低限度，所有样品的不断。的浓度，将需要任何新的（未知）复合调整，以达到所需的剂量响应范围。孵育10分钟，在25 ° C黑暗环境中。 </li><li>旋涡LUV样品，并加载到左边的注射器。装入正确的注射器钠缓冲或TL -淬灭。 </li><li>彻底清除气泡来回推注射器从两个样本。 </li><li>确保两个注射器加载之前关闭的阀门同样。不平等的加载会导致预混合的解决方案之前，他们到达录音室。 </li><li>第一个样本，记录荧光钠缓冲，重复4次，高电压（增益）进行必要的调整，重复5次。 </li><li>对于其余的样本，创纪录的9钠缓冲重复。 </li><li> TL -淬灭和创纪录的13重复更换钠缓冲。 </li><li>用清水冲洗，并继续进行下一个样品。作为对照，我们使用样本：与短杆菌肽和溶剂都没有用溶剂，没有最大的化合物的浓度和短杆菌肽，短杆菌肽。 </li></ol> 5。分析数据<ol><li>分析计算机传输的结果。阅读所有的MATLAB数据分析。对于每个样本，在所有的缓冲区和淬灭重复读，不包括在每种情况下的第一个，因为这些含有混合文物。假设120μL的驱动器体积略小于四杆，以彻底清除以前停流管混合物。 </li><li>所有样品的缓冲重复应该非常相似。如果有一个重大转变，在荧光信号，这取决于化合物的浓度，那么该化合物本身可能是荧光灯和它可能是必要的第一减去样品正常化之前，这个额外的荧光信号。 </li><li>手动经过的痕迹，清除“坏”的重复：重复多exponentiality由于混合文物和/或气泡中含有尖峰或偏差。 </li><li>规范化淬灭重复平均每个样品重复缓冲区的值。合并成一个可视化图形的所有样本的平均数。 </li><li>没有短杆菌肽记录的痕迹都应该相似，表明几乎没有荧光猝灭;有可能是一个缓慢的荧光信号，由于缓慢通过囊泡双层8 铊泄漏+减少。如果没有短杆菌肽和没有修饰符的示例显示了显着的淬火，然后水疱恶化，不应该再继续使用。如果没有短杆菌肽，但与修饰的样品表现出显着的淬火，然后修饰扰乱到这样的程度，它使荧光基团或淬灭跨双层囊泡双层。 </li><li>如果化合物改​​变脂质双分子层的属性，如短杆菌肽渠道检测，荧光的时间当然会明显改变。 </li><li>对于每个样本，拉伸指数<img src="/files/ftp_upload/2131/2131eg1.jpg" alt="公式1" />如4）是适合的第2-100毫秒归个人重复荧光猝灭曲线率<img src="/files/ftp_upload/2131/2131eg2.jpg" alt="图2" /> 4在2毫秒计算。一个给定的样本，从所有的个人重复计算平均值和标准偏差。 </li><li>最后，确定正常化的时间最接近的对照样品，短杆菌肽和无修饰的淬火率的相对变化。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/2131/2131fig1.jpg" alt="图1" /> 图1：要领的荧光猝灭为基础的检测，检测脂质双层属性的变化，左上：一个变焦单上用蚂蚁加在里面和在外面的NANO 3 ​​加TlNO 3纳米脂质囊泡。右上：（形成从上到下）使用无猝灭剂蝼蚁充满水泡，与淬灭剂与淬灭剂和87，260和780 nm的短杆菌肽预掺杂，记录荧光信号。底部：停流混合室的示意图。 <img src="/files/ftp_upload/2131/2131fig2.jpg" alt="图2" /> 图2：临数据SX软件的演示实验装置的描述中引用的各种面板的屏幕截图。 <img src="/files/ftp_upload/2131/2131fig3.jpg" alt="图3" /> 图3：多重复测定荧光信号从蚂蚁与钠缓冲加载LUVs第4个重复总是排除在外，因为它们包含混合文物。混合细胞样品注射器油管连接有一个定义的卷，因此最初的几个重复给我们读了什么是在以前的油管：重复1和2，结合一些水和样品重复3的水，大多样品重复4，为剩余的重复只是样品。 <img src="/files/ftp_upload/2131/2131fig4.jpg" alt="图4" /> 图4：多重复测定荧光信号从钠缓冲与TL -淬灭蚁装LUVs第4个重复已从两个条件。此外，TL -淬灭测量，重复8需要被删除，由于文物，最有可能的气泡。 <img src="/files/ftp_upload/2131/2131fig5.jpg" alt="图5" /> （一） 图5：辣椒素（CAP）对蚂蚁的荧光猝灭的时间当然归超过1秒的荧光信号，灰点表示所有重复的结果（N&gt; 5％的条件）;红色线条表示所有平均重复。 （二）第一个100毫秒，灰色点表示从单一重复的每个条件的结果;红色线条被拉长指数拟合（2 - 100毫秒），这些重复。点画蓝线表示的2毫秒大关，淬火率是确定的时间。在A和B的上部曲线显示结果没有铊+未来两年的痕迹显示在GA的情况下结果，与TL + ±章;低4跟踪显示与260 nm的GA和Tl +，其中结果数字表示，单位为μm[第]。为0，10，30和90微米的上限确定由一个拉伸指数率，淬火率分别为36 ± 6，69 ± 6，85 ± 8和247 ± 27（平均值± SD，N&gt; 8）分别。

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已提交临时专利申请，涉及的手稿中描述的方法。共同发明的HII和OSA。

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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<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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 ...

gramicidin-based-fluorescence-assay-for-determining-small-molecules

Research

JoVE Journal

Biology

12.7K 次观看.  Weill Cornell Medical College. 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。

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

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

科研

生物学

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

双层弹性性能的监测变化的单分子方法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

确定膜蛋白拓扑使用荧光蛋白酶保护（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

系绳双层脂膜，以监测金纳米粒子和脂膜之间的热传递

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Optical Clearing of Plant Tissues for Fluorescence Imaging

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In addition, autofluorescence attributed to chlorophyll hampers the observation of fluorescent proteins in plants. For a long time, various clearing reagents have been used to make plants transparent. However, conventional clearing reagents diminish fluorescent signals; therefore, it has not been possible to observe the cellular and intracellular structures with fluorescent proteins. Reagents were developed that can clear plant tissues by removing chlorophyll while maintaining fluorescent protein stability. A detailed protocol is provided here for the optical clearing of plant tissues using clearing reagents, ClearSee (CS) or ClearSeeAlpha (CSA). The preparation of cleared plant tissues involves three steps: fixation, washing, and clearing. Fixation is a crucial step in maintaining the cellular structures and intracellular stability of fluorescent proteins. The incubation time for clearing depends on the tissue type and species. In Arabidopsis thaliana, the time required for clearing with CS was 4 days for leaves and roots, 7 days for seedlings, and 1 month for pistils. CS also required a relatively short time of 4 days to make the gametophytic leaves of Physcomitrium patens transparent. In contrast, pistils in tobacco and torenia produced brown pigment due to oxidation during CS treatment. CSA reduced the brown pigment by preventing oxidation and could make tobacco and torenia pistils transparent, although it took a relatively long time (1 or 2 months). CS and CSA were also compatible with staining using chemical dyes, such as DAPI (4',6-diamidino-2-phenylindole) and Hoechst 33342 for DNA and Calcofluor White, SR2200, and Direct Red 23 for the cell wall. This method can be useful for whole-plant imaging to reveal intact morphology, developmental processes, plant-microbe interactions, and nematode infections.

Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning.

用于荧光成像的植物组织的光学清除

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In ...