nih-1r15hl13565-01 为 timothy j. lrocca 提供了支持

1. 准备缓冲器和试剂
注: 请参见表 1。
<ol><li>准备缓冲液 a、裂解缓冲液 b、样品缓冲液和数字宁的溶液。<ol>
<li>加入 8.77 g 的氯化钠和50毫升的 hepes (1 m, ph 7.4) 到900毫升去离子水, 用去离子水将最终体积调整为1升。 注: 最终浓度为 150 mm 氯化钠和 50 mm hepes。</li>
		<li>加入20毫升的 hepes (1 m, ph 7.4) 制备裂解缓冲液 b, kcl 0.75 g, mgcl2, 2 ml 乙二胺四乙酸 (0.5 m edta), 2 毫升乙二醇乙基乙基 (β-氨基乙醚)-n, n, n '-四乙酸 (0.5 m egta), 38.26 克甘露醇和23.96 克蔗糖至900毫升去离子水, 用去离子水将最终体积调整为1升。 注: 最终浓度为 20 mm hepes、10 mm kcl、2 mm mgcl 2、1mm edta、1 mm egta、210 mm 甘露醇和 70 mm 蔗糖。</li>
		<li>将 0.01 g 十二烷基硫酸钠 (sds) 加入10毫升的三缓冲盐水 (tbs), 最终浓度为 0.1% sds, 从而制备样品缓冲液。</li>
		<li>在去离子水的100毫升中加入25毫克的数字素 (最终浓度为 250μg/ml), 准备一个数字素的库存溶液。</li>
		<li>将所有缓冲液存储在 4°c, 将数字素存储在-20°c, 直到实验开始。</li>
	</ol>
</li>
	<li>准备新的蛋白酶和磷酸酶抑制剂的解决方案, 添加到缓冲溶液之前添加到细胞。<ol>
<li>将17.4 毫克的 pmsf 添加到100% 乙醇的1毫升 (最终浓度为 100 mm), 制备苯甲磺酰氟化硫 (pmsf) 的库存溶液。 注意: 在处理 pmsf 时, 请佩戴适当的防护设备并谨慎行事。私营军事和废物管理基金在摄入时具有危险性, 在皮肤接触 (刺激性)、眼神接触 (刺激性) 或吸入的情况下具有轻微危险;它对眼睛和皮肤有腐蚀性。</li>
		<li>根据制造商的说明准备一种市售的蛋白酶抑制剂鸡尾酒 (100x) (参见材料表)。</li>
		<li>在去离子水的1毫升中加入92毫克的 sov (最终浓度为 500 mm), 制备正奥瓦酸钠 (sov) 的库存溶液。 注意: 请佩戴适当的防护设备, 并在操作时小心操作。在眼睛接触 (刺激物)、摄入或吸入的情况下, sov 是危险的。严重的过度接触会导致死亡。</li>
	</ol>
</li>
</ol>2. 公共安全
<ol><li>在分馏前, 在磷酸盐缓冲盐水 (pbs) 中浓缩和清洗细胞。<ol>
<li>以适当的速度离心细胞悬浮液, 以形成颗粒。例如, 离心机将 u937 细胞悬浮在400xg 下 10分钟。</li>
		<li>取出上清液, 在室温 pbs 中重新悬浮细胞颗粒, 最终浓度为 4x106 cells/mL, 轻轻将移液器分解团块。</li>
		<li>以400xg 离心细胞悬浮液 10分钟以植入细胞。</li>
		<li>在最终浓度为 2x107 细胞的情况下, 取出上清液并在冰冷缓冲液 a 中重新悬浮细胞颗粒。 注: 所有后续步骤应在4°c或冰上执行, 所有缓冲器应预先冷却。</li>
	</ol>
</li>
</ol>3. 细胞蛋白分离
<ol><li>用洗涤剂的数字素孵育提取细胞质蛋白。<ol>
<li>紧接细胞重新悬浮之前 (步骤 3.1.3) 添加10μl 的库存永磁同步体 (100 mm), 10μl 蛋白酶抑制剂 (100x)、2μl 股票 sov (500 mm) 和100μl 的股票数尼宁 (250μg/ml) 至878μl 缓冲液 a (最终浓度为 1 mm pmsf), 1x 蛋白酶抑制剂, 1mm sov 和25μgml 数联宁;根据所使用的单元格数调整最终卷)。将溶液放在冰上, 直到细胞颗粒的加入。</li>
		<li>将细胞悬浮液在 400xg下离心 10分钟, 并去除上清液。</li>
		<li>在液相缓冲液中重新注射细胞颗粒含有抑制剂和数字素 (3.1.1 步骤制备) 的溶液, 最终浓度为 2x107 细胞, 轻轻地将移液器分解团块。</li>
		<li>在4°c 下将细胞悬浮液在端端旋转器上培养20分钟。</li>
		<li>将细胞悬浮液以400xg 离心 10分钟, 收集上清液, 并将其放入干净的离心管中。</li>
		<li>以 18, 000xg 离心收集的上清液 20分钟, 以颗粒细胞碎片。</li>
		<li>将上清液转移到干净的离心管中。</li>
		<li>重复步骤3.1.5 和 3.1.6, 直到离心后没有获得颗粒。</li>
		<li>收集含有细胞质蛋白的上清液, 并将其储存在 4°c (短期) 或-20°c (长期)。</li>
	</ol>
</li>
	<li>通过离心去除多余的数字蛋白和细胞质蛋白。<ol>
<li>在最终浓度为 4x10 6 细胞的情况下, 将具有数字渗透性的细胞颗粒 (从步骤 3.1.5)重新注入缓冲液 a 中, 轻轻将团块分解。</li>
		<li>将数字渗透电池悬浮液在 400xg下离心 10分钟, 并去除上清液。 注: 可以在缓冲液 a 中重复清洗, 以去除多余的细胞质污染物。</li>
	</ol>
</li>
</ol>4. 细胞同质化
<ol><li>将细胞在冰上的裂解缓冲液 b 中培养, 并通过机械方法裂解。<ol>
<li>在细胞重新悬浮之前 (步骤 4.1.2), 将10μl 的库存 pmsf (100 mm) 和2μl 的库存 sv (500 mm) 添加到988μl 的裂解缓冲液 b (最终浓度为 1 mm pmsf 和 1 mm sov; 调整最终体积, 以适应被裂解的细胞数量), 并保持独奏在冰上, 直到细胞颗粒的补充。</li>
		<li>在含有 pmsf 和 sov 的冰冷裂解缓冲液 b 溶液中, 在 14x106 细胞的最终浓度下, 将细胞颗粒 (从步骤 3.2.2) 重新沉淀。</li>
		<li>在冰上培养细胞悬浮液30分钟。</li>
		<li>将细胞悬浮液转移到冰上的预冷的盎司均质机 (带紧固的 b 穿山甲), 并使用缓慢、均匀的行程与均质机穿手一起执行40次传球。 注: 或者利用讨论部分详述的其他机械细胞裂解方法。</li>
		<li>收集均质, 并将其转移到一个干净的离心管。</li>
		<li>用小体积 (1 至2毫升) 的裂解缓冲液 b 清洗均质机的孔和管, 并将其添加到均质化体中。</li>
		<li>以 400xg (或颗粒未碎细胞所需的最小速度) 离心均匀度10分钟。</li>
		<li>将上清液转移到干净的离心管中。 注意: 如果一个重要的颗粒仍然重复步骤4.1.4 通过 4.1.6, 以提高分数的产量, 详见讨论部分。协议可以在这里暂停, 在4°c 下短期 (24小时) 储存。</li>
	</ol>
</li>
</ol>5. 差动离心
<ol><li>以越来越快的速度离心均质体, 以去除细胞碎片, 分离线粒体和膜组分。<ol>
<li>将上清液 (从步骤 4.1.8) 以500xg 离心 10分钟 , 将上清液转移到干净的离心管中, 并丢弃任何颗粒。</li>
		<li>以 1, 000x g离心上清液 (从步骤 5.1.1) 10分钟, 将上清液转移到干净的离心管中, 并丢弃任何颗粒。</li>
		<li>将上清液 (从步骤 5.1.2 )以 2, 000xg 离心 10分钟, 将上清液转移到干净的离心管中, 并丢弃任何颗粒。</li>
		<li>将上清液 (从步骤 5.1.3 )以 4, 000xg 离心 15分钟, 将上清液转移到干净的离心管中,保持颗粒中含有线粒体。</li>
		<li>在小体积 (0.5 \ e20120121 ml) 的裂解缓冲液 b 中再利用线粒体颗粒。</li>
		<li>以 4, 000xg 离心悬浮颗粒 15分钟 , 取出上清液, 并在所需的最后粒量样品缓冲液中重新悬浮线粒体颗粒 (例如, 250 至 500μl, 视颗粒的大小和所需的颗粒大小而定浓度)。</li>
		<li>将上清液 (从步骤 5.1.4) 以 4, 000xg 离心 15分钟, 将上清液转移到干净的离心管中。重复此步骤, 直到离心后没有获得颗粒。</li>
		<li>旋转上清液在 18, 000xg 3小时。</li>
		<li>去除上清液, 并保持颗粒含有膜蛋白。将膜颗粒重新循环到小体积 (0.5–1 ml) 的裂解缓冲液 b 中。</li>
		<li>以 18, 000xg 离心悬浮颗粒 1小时 。</li>
		<li>取出上清液, 并在所需的最终样品缓冲液体积 (250 至 500μl, 取决于颗粒的大小和所需的浓度) 中重新悬浮膜颗粒。</li>
	</ol>
</li>
	<li>在功率设置为 5 (20千赫的 125 w 最大功率的 50%, 见材料表) 的情况下, 在冰浴中对样品颗粒进行3秒的取样。</li>
	<li>将样品存放在 4°c (短期) 或-20°c (长期)。</li>
	<li>通过对细胞的细胞质、线粒体和膜隔间中的蛋白质标记物进行抗体检测样品的纯度 (参见具有代表性的结果部分)。</li>
</ol>

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提交人声明没有利益冲突

该方案的发展源于无法分离线粒体和膜样本, 使用商业上可获得的试剂盒, 以分析在坏死14期间的蛋白质定位.预制试剂盒的主要局限性是, 它们无法适应个别研究人员的需要, 每个样本的费用有限, 能够加工的样品数量有限。这里介绍的方法可以在不使用昂贵的试剂的情况下进行, 也不需要昂贵的设备。这种方法可以进行缩放, 以容纳任意数量的细胞, 并能够进行修改, 以适应研究人?...

在检查真核细胞中的分子途径时, 通常需要可靠地识别蛋白质定位。研究人员利用获得亚细胞组分的方法来更仔细地检查感兴趣的细胞成分。
大多数现有的细胞分馏方法一般分为两大类, 即基于洗涤剂的 1、2和基于超离心的3、4、5, 它们可以是以速度、精度和成本为区别。基于洗涤剂的协议依赖于使用具有不断增加的洗涤剂强度的缓冲液来溶解细胞的不同成分。这是一种快速方便的样品处理方法, 如果样品数量和大小较小, 则具有成本效益。以洗涤剂为基础的试剂盒可以从细胞中分离细胞质、膜细胞器 (混合组分) 和核组分。然而, 与这些试剂盒相关的一些缺点限制了他们对研究人员的有用性。它们旨在轻松分离单元的一个或两个组件, 但无法同时从样本中分离所有分数。洗涤剂的使用意味着质膜和膜封闭的细胞器将同样溶解, 因此, 无法相互分离。另一个复杂因素来自于这些试剂盒中的专有组件, 这些组件阻止研究人员更改特定应用的条件。最后, 它们的用途有限, 对于规模较大的实验来说可能费用高得令人望而却步。非洗涤剂为基础的试剂盒存在于线粒体的分离, 但是, 它们不是用来分离质膜的, 样品的收率明显低于密度离心分离协议6,7.
利用超离心获得分数的方法比基于洗涤剂的试剂盒更耗时, 但往往会产生更纯净的组分。要在不首先溶解细胞的情况下从细胞中分离等离子体膜 (导致膜细胞器污染), 需要用非洗涤剂方法对其进行裂解, 然后通过差异分离细胞成分离心--质膜分离需要 100, 000xg 的速度才能完成。在许多情况下, 差动离心之后必须进行等温密度梯度离心, 以进一步分离细胞组分或去除污染物。虽然这些方法是彻底和可修改的, 缺点包括成本, 时间消耗, 以及需要一个超离心分离的分数和进一步纯化通过密度梯度离心。大多数高速离心机的费用对个别调查人员来说是令人望而却步的, 而且往往是学术机构共享的核心设备。因此, 在这些情况下, 超离心机的可用性变得令人望而却步。
在这个分馏协议中, 我们证明了在不使用溶解性洗涤剂和不高速离心的情况下分离亚细胞分数。这种方法将使研究人员能够分离真核细胞的质膜、线粒体和细胞质成分, 并将分数之间的污染降至最低。

<table>
	<tbody>
		<tr>
			<td>Digitonin</td>
			<td>TCI Chemicals</td>
			<td>D0540</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>VWR</td>
			<td>22877-282</td>
			<td>For Homogenization</td>
		</tr>
		<tr>
			<td>end-over-end rotator</td>
			<td>Barnstead</td>
			<td>N/A</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td>Alfa Aesar</td>
			<td>J61721</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>GAPDH (14C10)</td>
			<td>Cell Signalling Technologies</td>
			<td>2118</td>
			<td>For detection of cytoplasmic fractions on western blot, dilution: 1:10000</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>J848</td>
			<td>For Lysis buffers A and B</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Alfa Aesar</td>
			<td>12315</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Na, K-ATPase a1 (D4Y7E)</td>
			<td>Cell Signalling Technologies</td>
			<td>23565</td>
			<td>For detection of plasma membrane fractions on western blot, dilution: 1:1000</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>793566</td>
			<td>For Lysis buffer A</td>
		</tr>
		<tr>
			<td>phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>VWR</td>
			<td>M145</td>
			<td>For Cytoplasm Extraction and Homogenization Buffer</td>
		</tr>
		<tr>
			<td>probe sonicator</td>
			<td>Qsonica</td>
			<td>Q125-110</td>
			<td>For Final Samples</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, General Use</td>
			<td>VWR</td>
			<td>M221-1ML</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>refrigerated centrifuge</td>
			<td>Beckman-Coulter</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>VWR</td>
			<td>227</td>
			<td>For Sample buffer</td>
		</tr>
		<tr>
			<td>sodium orthovanadate (SOV)</td>
			<td>Sigma-Aldrich</td>
			<td>450243</td>
			<td>For Lysis buffers A and B</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Tris-buffered Saline (TBS)</td>
			<td>VWR</td>
			<td>788</td>
			<td>For Sample buffer</td>
		</tr>
		<tr>
			<td>VDAC (D73D12)</td>
			<td>Cell Signalling Technologies</td>
			<td>4661</td>
			<td>For detection of mitochondrial fractions on western blot, dilution: 1:1000</td>
		</tr>
	</tbody>
</table>

cell fractionation of u937 cells in the absence of high-speed centrifugation

在该协议中, 我们详细介绍了在不使用超离心或不滥用洗涤剂的情况下获取 u937 细胞亚细胞分数的方法。该方法利用低渗缓冲液、数字素、机械裂解和差动离心分离细胞质、线粒体和质膜。这个过程可以扩大规模, 以满足研究人员的需求, 既便宜又直接。这种方法将使研究人员能够在没有专门离心机和商业试剂盒的情况下确定细胞中蛋白质的定位, 这两种方法的成本都高得令人望而却步。我们已经成功地使用这种方法分离人单细胞系 u937 中的细胞质、质膜和线粒体蛋白。

利用上面详细的协议和图 1所示的协议, 成功地完成了悬浮液中生长的未分化 u937 8 细胞的成功分馏。用这种方法获得的样品采用湿法转移到聚偏氟乙烯 (pvdf) 膜上进行了西方印迹9 。随后用细胞质、线粒体和膜局部蛋白标记抗体对膜进行了检测 (图 2,图 3,?...

Watch this Scientific Journal Video about Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation at JoVE.com

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

高速离心条件下 u937 细胞的细胞分裂

在这里, 我们提出了一个在不使用高速离心的情况下分离 u937 细胞的质膜、细胞质和线粒体的方案。该技术可用于纯化亚细胞组分, 以便随后通过免疫印迹检查蛋白质定位。

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

该协议允许研究人员获得细胞的不同分数，而无需使用专用设备、昂贵的试剂或复杂的技术。该协议的主要优点是技术的简单性，这使得它可修改和易于执行。演示这个程序的将是马特 · 德拉贡、罗布 · 哈卢斯卡和亚历克萨 · 霍奇斯。开始离心，将准备好的细胞悬浮液产生颗粒。拆下上一液，并在室温 PBS 中重新悬浮颗粒。将电池悬浮液以400 xg离心10分钟。然后去除上清液，在冰冷的缓冲液中重新悬浮细胞颗粒。根据文本协议准备缓冲区解决方案。将电池悬浮液在400 xg下离心10分钟，然后取出上一代。将缓冲液中的颗粒重新悬浮 A 溶液，最终浓度为每毫升 2 亿细胞，并轻轻移液器以分解任何团块。在此孵育后，细胞悬浮液在结束旋转器的端端上 4

cell-fractionation-u937-cells-absence-high-speed

Research

JoVE Journal

Biochemistry

13.5K 次观看.  Albany College of Pharmacy and Health Sciences. 该协议允许研究人员获得细胞的不同分数，而无需使用专用设备、昂贵的试剂或复杂的技术。该协议的主要优点是技术的简单性，这使得它可修改和易于执行。演示这个程序的将是马特 · 德拉贡、罗布 · 哈卢斯卡和亚历克萨 · 霍奇斯。开始离心，将准备好的细胞悬浮液产生颗粒。拆下上一液，并在室温 PBS 中重新悬浮颗粒。将电池悬浮液以400 xg离心10分钟。然后去除上清液?...

视频: Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

巨囊泡封装微球通过一个水包油乳液的离心制备

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

科研

生物化学

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em&gt;上皮细胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

氮气气蚀和差速离心允许监测外周血膜蛋白在细胞中的分布

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

可溶性和不溶性杭丁顿蛋白的分离分解

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.

使用免疫增强技术的超高速西布洛特

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...