This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1.准备的siRNA的解决方案<ol><li>重悬5纳摩尔BRCA2基因的siRNA干燥粒料加扰（Ctrl键）和5纳摩尔在100μl无RNase水（最终的siRNA浓度为50μM）。这是所述siRNA的库存，并且必须被储存在-20℃于10微升等分试样。 </li><li>取从所述siRNA库存10微升等分试样并加入无RNase水40微升，得到10μM的siRNA的工作溶液。该稀释必须存储在-20℃直到使用。 注：siRNA的工作溶液不应经受冷冻/解冻的3倍以上。 </li></ol> 2.播种人表皮细胞系<ol><li>在37℃，5％的CO 2从人上皮细胞系生长的细胞单层在组织培养板在培养箱中生长培养基中。 </li><li>洗细胞在板一次，用RT的PBS（10毫升的PBS为100毫米板）。 </li><li>以从板取下细胞，加入2ml的0.25％胰蛋白酶的/ 0.53毫摩尔EDTA溶液孵育3-5分钟取决于细胞类型。 </li><li>之前收集分离的细胞中，加入2ml含10％胎牛血清的完全培养基的中和胰蛋白酶。 </li><li>转移细胞到15毫升管。 </li><li>在吊桶式转子离心细胞在320-330×g离心3分钟，在4℃。 </li><li>重悬细胞沉淀在5ml抗生素的培养基中。 </li><li>算在Burker室中的显微镜下或通过根据制造商的说明使用自动计数系统的细胞。 </li><li>种子细胞在6孔板24小时（0.5-1×10 6个细胞在2ml培养基的每孔）后为约80％汇合。最佳细胞数接种可以取决于特定细胞系和生长速度各不相同。 注意：不要在中在这一点上添加抗生素，因为抗生素会负transfe的干扰效率ction。这是很重要的，该细胞是在低培养传代（&lt;10），以实现BRCA2 siRNA转染的高效率。 </li></ol> 3.转染细胞的siRNA <ol><li>电镀细胞后二十四小时内，继续进行第一轮的转染。 <ol><li>稀释6微升基于脂质的10特异性的siRNA转染试剂在130微升减少血清培养基。 </li><li>在130微升减少血清培养基稀释3微升10μM的siRNA（30皮摩尔）。 </li><li>结合稀释的siRNA与稀释的转染试剂，孵育在室温5-10分钟。 </li><li>在温育后，除去从细胞培养基中，并添加新鲜培养基预先加热至37℃（无抗生素）2毫升</li><li>加入250微升的siRNA转染试剂复合物的细胞（该量是用于一个6孔板的单个孔）。 </li><li>孵育细胞在加湿培养箱中在3776下用5％的CO 2。 </li></ol></li><li> 24小时后，继续进行第二轮的转染重复步骤3.1.1-3.1.5并作以下修改为3.1.2步：稀释5微升10μM的siRNA（50皮摩尔）的130微升减少血清培养基。 <ol><li>分析前孵育细胞1-2天，在37℃。 注：两轮转染和使用在第二轮高的siRNA /转染试剂的比例是必不可少得到BRCA2沉默的效率高。 </li></ol></li></ol> 4.裂解细胞免疫分析<ol><li>制备新鲜的裂解缓冲液与含有非离子型，非变性去污剂辛基苯氧基的1％，5毫焦磷酸钠，1mM的原钒酸钠，10mM的氟化钠，2mM的苯基甲基磺酰氟，10微克的PBS（pH为7.4）/ ml亮肽素，10微克/ ml抑肽酶。保持它在冰上。 </li><li>除去从细胞以及w的介质用冷PBS（2毫升/孔）在该板一次灰它们。为了防止交叉污染的PBS，它完全从板块中删除。 </li><li>加入200μl裂解缓冲液到每个孔中。 注意：如果该细胞必须也用于其他测定法，trypsinize细胞如步骤2.1-2.6中描述和悬浮细胞中根据应用适当的缓冲/介质中使用。 </li><li>轻轻转动该板均匀地分布在裂解缓冲液孵育15分钟，在4℃下在旋转器上。 </li><li>收集在使用细胞刮刀冰离心管的细胞裂解物。 </li><li>离心机在17900×g离心25分钟，在4℃，并收集在一个新的管中的上清液。 </li><li>测量使用根据制造商的说明市售蛋白质测定蛋白质浓度。 </li></ol> 5. BRCA2免疫印迹分析<ol><li>通过稀释加入50ml 20×Tris-醋酸SDS电泳b制备运行缓冲uffer（50毫曲辛，的50mM Tris碱，0.1％SDS，pH值8.24）950毫升蒸馏水。 </li><li>制备的样品如下：添加15微克总细胞裂解物，将5μl的4倍样品缓冲液含有十二烷基硫酸锂，在pH 8.4，加入2μl10×样本还原剂（0.5M DTT），和裂解缓冲液至20中的最终体积微升。 注意：此协议允许检测BRCA2的正常细胞系也当使用总蛋白的较低量（降至5微克）。 </li><li>变性的样品在55℃下进行10分钟。 注：关键是要使用55℃。因为BRCA2蛋白是热敏性11，更高的变性温度导致完整的全长（390 kDa）的BRCA2蛋白一致损失。 </li><li>根据制造商的说明建立的电泳室。 </li><li>装载样品和10微升的高分子量预染色的蛋白质标准进行预制凝胶（Tris-醋酸3-8％），并在运行120伏的AB出2小时12。停止55 kDa的蓝色标记离开预制胶前的电泳运行。 </li><li>准备转移缓冲液用50毫升20×转移缓冲液，加入100毫升100％甲醇和850毫升水中。 </li><li>激活PVDF膜（0.45微米孔径）通过浸泡在100％甲醇5分钟，用蒸馏水漂洗并平衡在转移缓冲液至少5分钟。浸泡在转移缓冲液传送装置的海绵，在4℃直至使用。 </li><li>当电泳运行结束，组装在以下顺序（自底向上）传输堆栈夹层：在转移缓冲液饱和3海绵一3MM级纸张饱和在转移缓冲液，凝胶，活化PVDF膜，人们3MM在转移缓冲液饱和级纸，三海绵饱和传输缓冲区13,14。放置在栈入装置和转移蛋白在350mA 4小时，在4℃或在180毫安过夜，在4℃。 注意：存在BRCA2一个非常大的蛋白质，它是非常重要的，从1小时延长转印时间（所建议的制造商，大多数传输协议），以4小时或过夜，以确保高的分子量的蛋白质的完整的传输。另外，由于长期的传输时间，有必要在4℃下进行杂交在冷室中，以尽量减少过热和促进三明治拆卸。半干转移系统是不利于传输大量的蛋白质。 </li><li>转移后，用TBS洗膜，阻止它为1小时，在RT在TBS-吐温0.25％（TBS-T）含有5％脱脂奶粉和探针过夜，在4℃下用30微升抗BRCA2兔多克隆抗体（ 200微克/毫升），稀释于9ml TBS-T + 5％脱脂奶粉[1：300（体积/体积）稀释。 </li><li>用TBS-T洗膜三次（每次10分钟），然后孵育的过滤器1小时，用1微升辣根过氧化物酶缀合的抗兔次级抗体稀释在10的TBS毫升-T + 5％脱脂奶粉。此后，用TBS-T洗膜三次（每次10分钟）。 </li><li>通过ECL进行免疫检测使用增强的化学发光（ECL）的Western印迹检测试剂，按照制造商的说明来揭示BRCA2蛋白。可视化高分辨率ECL电影immunochemiluminescent带。 </li><li>在10 1000（10微升抗微管蛋白抗体的制备：与抗体通过使用单克隆抗体与β微管蛋白稀释在1执行免疫检测对于持家基因，如β微管蛋白（55 kDa）的，在相同的过滤器作为负载对照毫升TBS-T + 5％无脂干牛奶）中1小时，在室温。洗和孵化如对于BRCA2（5.10-5.11）第二抗体，除了使用辣根过氧化物酶缀合的抗小鼠代替抗兔二级抗体。 </li><li>获得与immunochemiluminescent频带印象的膜的数字图像并分析由专用的图像分析软件的谱带强度（ 如，ImageJ的）。 </li></ol>

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The authors have nothing to disclose.

由于BRCA2基因导致几个癌症类型，包括女性和男性乳腺癌，卵巢癌，前列腺癌，胰腺癌和黑素瘤3,4-风险增加的种系突变，一些研究已经进行了解的生物学功能在BRCA2蛋白质。大部分这些研究是基于基因主要是由于在分析像BRCA2巨蛋白的技术困难。此处描述的用于沉默和分析BRCA2蛋白表达的方法，提供了有效的工具来调查BRCA2肿瘤抑制基因的功能的生物测定的广谱。沉默协议是基...

在BRCA2（乳房癌易感基因-2）基因编码肿瘤抑制蛋白，发挥在通过调节重组酶的Rad51 1的功能修复DNA双链断裂了至关重要的作用。 BRCA2也被牵连于转录的调节和细胞周期调控2。在BRCA2基因的种系突变诱导的常染色体显性遗传易感性乳腺癌和在妇女卵巢癌和前列腺癌的男性，以及其他类型的癌症3,4-倾向。然而，尽管在发展癌症，BRCA2基因突变是抑制或减少BRCA2功能可能使肿瘤细胞更容易受到化疗剂引起的DNA损伤5-7的风险增加。零星的癌症表现出BRCA2基因突变（&lt;3％），尽管BRCA2蛋白水平降低已在一些癌症类型被检测到低速率，这表明BRCA2蛋白通过非突变依赖性机制-7,8-肿瘤发生过程中可能会丢失在零星的癌症。因此，要充分认识BRCA2功能在癌症生物学的上下文中以及在其他生物的设置是重​​要的。 用于鉴定在哺乳动物细胞中的基因的新颖功能的强大的工具是沉默其表达。作为一个例子，在各种正常上皮细胞的沉默BRCA2的表达最近导致了新颖BRCA2作为失巢凋亡抗性，采集的癌细胞侵入和转移能力9中的一个重要步骤调 ​​节器的功能的识别。基因沉默可以通过引入细胞中任一小干扰（SI）RNA或短发夹（sh的）RNA分子靶向特定基因来实现。 siRNA的高效力和它的易用性使得它的优先工具沉默实验旨在获得新的见解关键的生物进程第二，以确定新的治疗目标。然而，基因表达的有效的敲低可能并不容易实现对所有的基因，可能是高度可变的取决于细胞类型。因为在细胞内蛋白质水平的降低是所研究的最相关的表型，这是至关重要的免疫印迹的基因产物的分析以量化基因沉默。与这方面，BRCA2蛋白呈现了进一步的挑战：作为一个大的蛋白质（约390 kDa的），技术困难确实存在对常规的生化分析，包括免疫印迹。 我们在这里报告BRCA2在人类上皮细胞系的高效沉默和迅速和成功的检测用免疫印迹法击倒BRCA2蛋白优化的协议。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BRCA2 siRNA</td>
			<td>Dharmacon</td>
			<td>L-003462-00</td>
			<td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td>
		</tr>
		<tr>
			<td>Ctrl siRNA</td>
			<td>Dharmacon</td>
			<td>&#160;D-001810-10-05</td>
			<td>ON-TARGETplus Non-targeting Pool</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Reagent&#160;</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/13778075">13778075</a></td>
			<td>Transfection Reagent</td>
		</tr>
		<tr>
			<td>OPTI-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Medium for transfection procedure</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td>
			<td>Life Technologies</td>
			<td>EA0378</td>
			<td>Gel for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample buffer&#160;</td>
			<td>Life Technologies</td>
			<td>NP0007</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Sample Reducing agent (10x)</td>
			<td>Life Technologies</td>
			<td>NP0004</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Antioxidant</td>
			<td>Life Technologies</td>
			<td>NP0005</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>HiMark Pre-stained protein standard</td>
			<td>Life Technologies</td>
			<td>LC5699</td>
			<td>Prestained marker for gel electrophoresis</td>
		</tr>
		<tr>
			<td>XCell SureLoc Mini-Cell&#160; System</td>
			<td>Life Technologies</td>
			<td>EI0002</td>
			<td>Equipment for protein electrophoresis/transfer</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate SDS Running Buffer</td>
			<td>Life Technologies</td>
			<td>LA0041</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Life Technologies</td>
			<td>NP0006-1</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>BRCA2 H-300 rabbit polyclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8326</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Beta-tubulin monoclonal antibody</td>
			<td>Sigma Aldrich</td>
			<td>T4026-100UL</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Skim Milk powder</td>
			<td>Sigma Aldrich</td>
			<td>70166</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td>
			<td>Pierce Thermo Scientific</td>
			<td>34080/34096</td>
			<td>Chemiluminescence system</td>
		</tr>
		<tr>
			<td>PNT1A cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>95012614</td>
			<td>PNT1A human, normal prostate epithelium immortalized with SV40</td>
		</tr>
		<tr>
			<td>Nthy cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>90011609</td>
			<td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td>
		</tr>
		<tr>
			<td>Cell Proliferation Kit I (MTT)</td>
			<td>Roche</td>
			<td>11465007001</td>
			<td>Kit for cell proliferation assays</td>
		</tr>
	</tbody>
</table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

肿瘤抑制蛋白BRCA2及其检测常规生化分析的沉默代表了由于人类BRCA2蛋白（约390 kDa的）的大尺寸巨大的技术挑战。我们报告的标准siRNA转染和免疫印迹协议的修改来沉默人类BRCA2和检测内源性BRCA2蛋白，分别在人上皮细胞系。关键的步骤包括一个高的siRNA转染试剂的比例和相同的实验中的两个随后的几轮siRNA转染的。使用这些和其他的修改标准协议中，我们一致地实现人类BRCA2基因的70％以上的沉默作为判断免疫印迹分析用抗BRCA2抗体。此外，细胞裂解物在55℃，而不是传统的70-100℃，并在免疫印迹程序的其他技术的优化变性允许检测完好BRCA2蛋白偶的wh烯极低量原料可用或当BRCA2蛋白质表达水平非常低。 BRCA2的人类细胞中沉默效率提供了一个有价值的策略，以扰乱BRCA2功能的细胞中具有完整BRCA2，包括肿瘤细胞，以检查新的分子途径，并且可能会受致病BRCA2基因突变的肿瘤中的细胞功能。此协议为有效的沉默等“大”蛋白如BRCA2的分析，适应应该是很容易实现的。

在继续进行的生物/生化测定来检查BRCA2沉默对细胞功能的影响，第一个步骤是通过使用一个加扰的siRNA（非靶向siRNA）并排BRCA2的siRNA（ 图1A确认沉默的特异性）。它执行第二轮的siRNA转染的具有高的siRNA /转染率， ​​以得到BRCA2沉默的高效率是非常重要的。实际上，仅执行一个回合siRNA转染的或用在第二轮（25皮摩尔的siRNA和6微升转染试剂）低级的siRNA转染的比例会导致低...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

沉默<em&gt; BRCA2</em&gt;以确定新的BRCA2调控的生物学功能在人体细胞培养

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

silencing-brca2-to-identify-novel-brca2-regulated-biological

Research

JoVE Journal

Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

9.0K 次观看.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 

视频: Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

在原代培养海马神经元的密集核心囊泡的实时成像

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

科研

生物学

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

葡萄糖摄取测量和对胰岛素刺激的反应<em&gt;体外</em&gt;培养的人类初级肌管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...