This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

小鼠实验程序批准了麦吉尔大学（协议4966）的动物护理委员会。 1.准备从培养细胞蛋白提取物<ol><li>用10毫升冰冷的磷酸盐缓冲盐水（PBS）洗两次培养的细胞。 </li><li>刮贴壁细胞与任一橡皮或塑料细胞刮棒在1ml冰冷的PBS，转印悬浮到1.5ml微量离心管中。 </li><li>旋在微量离心5分钟，于700×g离心，于4℃。吸PBS。 </li><li>加入100微升冰冷的细胞质裂解缓冲液（ 见表1）每10 7个细胞，并吸取向上和向下。 </li><li>在冰上孵育20分钟。 </li><li>旋，在4℃下以全速10分钟在微量。 </li><li>丢弃沉淀。上清转移到新的1.5 ml离心管，并保持在冰上。 </li><li> DET使用Bradford法23 - （10微克/微升通常1）貂皮蛋白浓度。 </li><li>等分试样并储存细胞提取物在-80℃下直到使用。 </li></ol> 2.准备从小鼠肝脏和脾脏蛋白提取物<ol><li>安乐死鼠标使用CO 2吸入。 </li><li>奠定安乐死的动物在一块干净的纱布在解剖板。打开用剪刀腹部。 </li><li>解剖肝脏和脾脏用剪刀和镊子，并冲洗在大约50毫升的冰冷的PBS各组织。 </li><li>立即切割组织切成小块，用手术刀（例如:约1 - 2 立方毫米）。 </li><li>无延迟，放在新鲜冻存管组织的片，然后捕捉冻结他们在液氮。商店管理单元冷冻组织等分在-80℃下直到使用。 </li><li>在0.25 - （2 mm 3的大约1） - 0.5毫升匀化一片冷冻组织的冰冷细胞质裂解缓冲液（ 见表1）用组织匀浆10秒。 </li><li>转移匀浆至1.5 ml离心管，并冰浴20分钟。 </li><li>旋，在4℃下以全速10分钟在微量。 </li><li>放弃颗粒和转移上清到新的1.5 ml离心管。保持在冰上。 </li><li>测定蛋白浓度-使用Bradford测定法23（通常为1 10微克/微升）。 </li><li>等分试样并储存细胞提取物在-80℃下直到使用。 </li></ol> 3.准备放射性标记IRE-探头<ol><li>通过在37℃下用限制性核酸内切酶XbaⅠ位（每质粒微克1 U）的IRE序列的下游其裂解温育1小时进行线性化的含有质粒IRE I-12.CAT 22。将线性化质粒将被用作模板的体外转录。 </li><li>设置一个中的 n 体外在20微升的总体积的转录反应。使用表2中所示的储液，并添加1微升线性质粒模板，4微升的转录缓冲液1微升混合物中ATP / CTP / GTP混合，加入10μl[α-32 p] -UTP，2微升二硫苏糖醇，1微升RNA酶抑制剂和1μlT7 RNA聚合酶。通过上下吹打混匀。 </li><li>孵育在40℃下1小时24。 </li></ol> 4.净化放射性标记IRE-探头<ol><li>加入1微升的0.5M EDTA，pH为8混音通过上下抽吸终止的体外转录反应。 </li><li>添加10微升10毫克/毫升的tRNA，作为载体更好沉淀。通过上下吹打混匀。 </li><li>添加82.5微升的3M乙酸铵。涡旋混合。 </li><li>添加273微升乙醇。涡旋混合。 </li><li>静置在室温5分钟。 </li><li>在微型在RT旋转10分钟以全速。弃去上清液。 </li><li>洗涤沉淀用100μl的70％乙醇。 </li><li>在微型在RT旋转10分钟以全速。弃去上清液。 </li><li>空气干燥沉淀10分钟。 </li><li>在100μl双蒸重悬沉淀，预先高压灭菌的 H 2 O. </li><li>量化放射性用液体闪烁计数器，等分试样的放射性标记的探针的IRE并储存在-80℃直到使用。冷冻等分试样可用于长达3周。 </li></ol> 5.准备EMSA原生聚丙烯酰胺凝胶<ol><li>通过使用1.5毫米垫片和梳组装凝胶（16×16厘米）。 </li><li>以制备6％非变性聚丙烯酰胺凝胶，使用表3中所示的储备溶液混合7.5毫升40％的丙烯酰胺:双丙烯酰胺，加入5ml 5×TBE和37.5毫升双蒸H 2 O. </li><li>加入0.5毫升10％的新制备的过硫酸铵（APS）和25微升四甲基乙二胺（TEMED）。 </li><li>立即倒入丙烯酰胺溶液的凝胶，并让它聚合。等待大约30分钟。 </li><li>与凝胶组装电泳装置中，填充罐用0.5X TBE并连接电源。 </li></ol> 6.凝胶电泳迁移分析<ol><li>在10微升的总体积稀释25微克从细胞或组织与细胞质裂解缓冲液（ 表1）的蛋白提取物（低蛋白质浓度也可以使用）。保持在冰上。如果需要的话，加入1微升1:4稀释的2-巯基乙醇（2-ME），以激活休眠IRP1（终浓度:2％）25。 </li><li>稀释放射性IRE探头双蒸H 2 O来200000 CPM /μL，热在95℃变性℃1分钟，并冷却在RT至少5分钟。 </li><li>成立EMSA反应通过加入1微升放射性IRE探针与蛋白质提取物。 </li><li>孵育20分钟，在RT。 </li><li>加入1微升50毫克/毫升肝素的反应（以抑制非特异性蛋白相互作用与探针9），并继续孵育另外10分钟。分装肝素在-80℃下储备溶液（50毫克/毫升），并存储。 </li><li>当使用放射性标记的长探针（&gt; 60个核苷酸），加入1微升RNA酶T1（1U /微升），孵育10分钟，在室温，以减少非特异性蛋白结合到探针，并允许更好地分离的RNA /蛋白质的电泳过程中的复杂。 </li><li>加入3微升加样缓冲液（80％甘油+溴酚蓝），混合和负载上的6％非变性聚丙烯酰胺凝胶。 </li><li>运行凝胶在130 V（5伏/厘米）60分钟。 </li><li>牛逼转输的凝胶到大滤纸和干燥。 </li><li>暴露于膜和开发放射自显影。暴露时间的范围可以从1小时（或更少）至O / N。 </li></ol>

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The authors declare that they have no competing financial interests.

在此，我们描述了已开发研究IRP1和IRP2的IRE结合活动的协议，并且我们显示代表性数据。通过使用不同的探针，该协议也可以被用于其它RNA结合蛋白的研究调整。一个关键步骤是探针的大小。用法长探针，这是共同的时，确切的结合位点是未知的，可能会导致的RNA /蛋白质复合物不不同迁移比游离的RNA。在这种情况下，最好是通过用RNA酶T1（ 步骤6.6）处理以去除未结合的RNA。探针的质?...

在EMSA最初被开发，研究DNA结合蛋白与靶DNA序列1,2的关联。其原理是类似的RNA /蛋白质相互作用3，这是本文的重点。简言之，RNA被带负电荷并朝向阳极期间非变性电泳聚丙烯酰胺（或琼脂糖）凝胶将迁移。凝胶内的迁移取决于核糖核酸，其正比于它的电荷的大小。相比于自由RNA特异性的蛋白质的RNA的结合改变其流动性，且复杂的迁移变慢。这主要是由于增加的分子量，而且要在电荷和可能的构象的改变。利用RNA标记为探针，可以方便地监控了"凝胶阻滞"或"条带迁移"的。的32 P标记的RNA探针的使用是很常见的，并提供高灵敏度。被检测的RNA /蛋白质复合物和游离RNA的迁移通过放射自显影。缺点是:32 P（14.29天），探针的质量由于辐逐渐恶化的半衰期短，一个放射性许可证和基础设施为放射性工作，以及潜在的生物安全的担忧的要求。因此，已经开发了替代非同位素方法用于标记的RNA探针，例如用荧光团或生物素，其能够检测由荧光或化学发光成像4,5。这些方法的局限性是成本较高并且经常敏感性降低相比同位素标记和非同位素标记物的潜在干扰的RNA /蛋白质相互作用。非变性聚丙烯酰胺凝胶适用于大多数应用EMSA和常用。有时，琼脂糖凝胶可能造成的大型复合物的分析，另一种选择。 EMSA的主要优点在于，它结合了简单，灵敏度和鲁棒性4 &lt;/ SUP&gt;。该测定可以在几个小时内完成，并且不需要复杂的仪器。 RNA /蛋白质相互作用可通过EMSA在浓度低至0.1纳米或更小进行检测，并广泛的结合条件内（pH为4.0 - 9.5，单价盐浓度1 - 300毫米，而温度0 - 60℃）。 RNA /蛋白质复合物的形成，也可以由滤波器结合测定研究。这是基于对RNA /蛋白质复合物中的硝化纤维过滤器中的保留一个简单，快速，和便宜的程序，而自由RNA探针穿过6。相比EMSA，它的情况下将RNA探针含有多个结合位点，或粗提取物包含一个以上的RNA结合蛋白结合的探针在同一地点的限制。而多的RNA /蛋白质相互作用会逃过检测由滤波器结合测定中，它们可以容易地通过EMSA观察。在某些情况下，可视化前夕n个可能时两个RNA /蛋白质复合物共迁移（例如，人类IRP1 / IRE和IRP2 / IRE复合物）中，加入的抗体对RNA结合蛋白的EMSA反应之一，在凝胶上产生进一步的延迟（ "超迁移"）7。 在EMSA已广泛用于研究IRP1和IRP2，这是铁代谢8-10转录后调节器。它们的工作由几个11的mRNA的非编码区中结合IRES，系统发育保守的发夹结构。 IRE有时首先在编码铁蛋白12和转铁蛋白受体1（TFR1）13，铁贮存和吸收，分别为蛋白质的mRNA的发现。稍后，IRE有时被发现在红细胞特异性氨基乙酰丙酸合成酶 （ALAS2）14，线粒体乌头15，铁转运蛋白16， 二价金属转运蛋白1（DMT1）17，缺氧诱导因子2 <e米&gt;α（HIF2α）18，以及其他的mRNA 19-21。原型H和L-铁的mRNA包含在其5 1 IRE&#39;UTR，而TFR1基因包含在其3多IRE有时&#39;UTR。 IRE / IRP的相互作用特异性抑制铁蛋白mRNA的翻译在空间上阻断43S核糖体亚基及其相关;此外，他们稳定TFR1反对内切裂解的mRNA。 IRP1和IRP2份额广泛的序列相似性，并表现出铁饥饿细胞高IRE结合活性。在铁充足的细胞，IRP1组装转换它到胞质乌头在其IRE结合活性为代价一立方烷的Fe-S簇，而IRP2经历蛋白酶体降解。因此，IRE / IRP相互作用取决于蜂窝铁状态，但也受其它信号，如H 2 O 2，一氧化氮（NO）或缺氧。在这里，我们描述了协议，以评估IRE-结合活性从原油细胞和组织的提取物用EMSA。我们使用的是通过体外转录从质粒DNA模板（I-12.CAT），其中IRE序列由最初引入有义方向的T7 RNA聚合酶位点下游生成一个32 P-标记的H-铁蛋白的IRE探针克隆退火合成寡核苷酸22。

<table border="0" cellpadding="0" cellspacing="0" width="430">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:195px;">Name of the Materials&#160;</td>
			<td style="width:119px;">Company</td>
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		<tr height="17">
			<td height="17" style="height:17px;">leupeptin</td>
			<td>SIGMA</td>
			<td>L2884</td>
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			<td height="17" style="height:17px;">PMSF</td>
			<td>SIGMA</td>
			<td align="right">78830</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BioRad Protein Assay</td>
			<td>BIORAD</td>
			<td>500-0006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T7 RNA polymerase</td>
			<td>Thermoscientific</td>
			<td>EPO111</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase Inhibitor</td>
			<td>Invitrogen</td>
			<td>15518-012</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">UTP [alpha-32P]</td>
			<td>Perkin-Elmer</td>
			<td>NEG507H</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation liquid</td>
			<td>Beckman Coulter</td>
			<td align="right">141349</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean II xi Cell&#160;&#160;</td>
			<td>BIORAD</td>
			<td>165-1834</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
		</tr>
	</tbody>
</table>

electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

RNA /蛋白质相互作用是转录后调控途径的关键。间的最佳表征的胞质RNA结合蛋白是铁调节蛋白 ，IRP1和IRP2。它们结合到铁几个靶mRNA的非翻译区（UTR）内响应元件（IRES），从而控制的mRNA翻译或稳定性。 IRE / IRP的相互作用已被广泛研究了EMSA。在这里，我们描述了EMSA协议用于分析IRP1和IRP2的IRE结合活性，这可以推广到其他评估RNA结合蛋白的活性。含有一种RNA结合蛋白，或纯化此蛋白质的制备的粗蛋白裂解物，孵育用过量的32 P标记的RNA探针，从而允许形成复合物。肝素加入到排除非特异性蛋白探查结合。随后，将混合物通过在聚丙烯酰胺凝胶非变性电泳分析。免费探头迁移速度快，而所述RNA /蛋白质复合物表现出延迟的流动性;因此，该过程也被称为"凝胶阻滞"或"条带迁移"测定法。电泳结束后，将凝胶干燥和RNA /蛋白质复合物，以及游离的探针，通过放射自显影检测。该协议的总体目标是检测和量化的IRE / IRP和其它RNA /蛋白质相互作用。此外，EMSA也可用于确定特异性所研究的RNA /蛋白质相互作用，结合亲和力和化学计量。

放射标记的IRE探针制备，如在第3和第4的协议的说明。所述探针的序列为5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUGÇUUCAAÇAGUGC UUGGA CGGAUCCU-3&#39;;粗体的核苷酸代表一个未配对的C残基和环，这是至关重要的IRE特征。探针的比放射性为4.5×10 9的cpm / RNA微克。 评估铁扰动对IRE结合活性的影响，鼠的RAW264.7巨噬细胞不进行治疗，或者与血红素（铁源）或去铁胺（铁螯合剂）...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

在这里，我们提出了一个协议分析RNA /蛋白质相互作用。电泳迁移实验（EMSA）是基于RNA的/蛋白复合物和游离RNA在天然凝胶电泳迁移差异上。通过使用放射性标记的RNA探针，RNA /蛋白质复合物可以通过放射自显影进行显现。

凝胶电泳迁移分析（EMSA）为RNA，蛋白质相互作用的研究:IRE / IRP示例

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.5K 次观看.  Jewish General Hospital. 在这里，我们提出了一个协议分析RNA /蛋白质相互作用。电泳迁移实验（EMSA）是基于RNA的/蛋白复合物和游离RNA在天然凝胶电泳迁移差异上。通过使用放射性标记的RNA探针，RNA /蛋白质复合物可以通过放射自显影进行显现。 

视频: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

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

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

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

测试的速度和运动方向的视觉灵敏度在蜥蜴

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

科研

生物学

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

液相亲和捕获试验用磁珠研究蛋白质相互作用:脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

凝胶电泳迁移分析（EMSA）为RNA，蛋白质相互作用的研究:IRE / IRP示例

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