这项工作得到了 NIH (RO1-NS090390) 的支持。我们还要感谢琼 Steffanfor 博士在这项试验的开发过程中提供的技术援助和讨论。

动物伦理学声明-实验是严格按照《国家卫生研究院的实验室动物的护理和使用指南》和机构动物保育和使用委员会批准的动物研究议定书进行的 (IACUC) 在加利福尼亚大学欧文分校, AAALAC 认证机构。尽一切努力尽量减少动物的痛苦。
1. 裂解缓冲液的制备
<ol><li>通过150µm 过滤器制备 "可溶性" 裂解缓冲液 (10 毫米三 pH 7.4, 1% 个海卫 x 100, 10% 毫米氯化钠, 0.22 甘油) 和无菌过滤器。<ol>
<li>对于一个工作的 "可溶性" 溶解缓冲, 测量 n-Ethylmaleimide (NEM) 和彻底溶解在100毫米 phenylmethysulfonyl 氟 (PMSF) 溶解在100% 乙醇。 注: 裂解缓冲抑制剂: (i.) 20 毫米 n-ethylmaleimide (NEM);(二) 0.2 毫米 PMSF;(iii) 1 毫米原钒酸钠 (Na3VO4);(iv) 1 µg/毫升 leupeptin;(v) 1 µg/毫升抑肽酶;(vi) 20 毫米氟化钠 (NaF)。 注意: 在整个协议中使用 NEM 时, 戴口罩。必须使工作裂解缓冲液保持新鲜, 以确保活性抑制剂。</li>
		<li>对于具有蛋白酶抑制剂的可溶性溶解缓冲剂, 在以下顺序添加抑制剂: 氟化钠 (NaF), 高达容积与冷裂解缓冲, 原钒酸钠 (Na3VO4), 抑肽酶和 Leupeptin。</li>
	</ol>
</li>
	<li>对于 "不溶性" 溶解缓冲, 补充 4% sds, 增加500µL 20% sds 到2毫升的 "可溶性" 溶解缓冲。在室温下离开。 注: SDS 将沉淀, 当保持在4摄氏度;把这个留在室温下。最后溶解缓冲成分:10 毫米三 (pH 7.4);1% 海卫 X-100;150毫米氯化钠;10% 甘油;4% SDS (仅不溶)。</li>
</ol>2. 可溶性/不溶性分馏协议
注意: 有关示意图, 请参见图 1 。
<ol><li>标签两套管为每个样品: "可溶性" 和 "不溶性"。</li>
	<li>添加µL 的冰冷工作可溶性溶解缓冲剂, 补充蛋白酶抑制剂 (见 1.1.1) 组织。 注: X = 体积随组织量的不同而变化;请参见表 1。</li>
	<li>对于小鼠脑组织 (即, 纹状体), dounce 缓慢30倍, 在玻璃1毫升 douncer 和吸管成 "不溶" 标记管 (保持冰)。 注: 在启动 douncing 后要小心不要打破液体表面, 因为气泡会形成, 可能产生不完全均匀化。</li>
	<li>对于细胞 (即, HeLa, HEK293T), 用冷的 1x PBS 冲洗一次细胞。移除 PBS 并将最小的溶解缓冲量应用到细胞中, 用细胞刮刀提升。 注: 对于上述细胞线, 细胞被镀在 5 x 105细胞每6个井板和生长到接近融合。一个井相当于小鼠纹状体的体积。</li>
	<li>将匀浆或细胞裂解物转化为标签, "不溶性" 管和溶解在冰 1 h (开始时钟后, 最终管处理)。 注: 对于细胞裂解物, triturate 多次在开始孵化前分解细胞团簇。避免气泡的形成。简要地漩涡脉冲每个样品为 1 s 中途通过株溶藻。</li>
	<li>离心机样品在4°c 在 1.5万 x g 为20分钟。</li>
	<li>恢复上清为 "可溶性" 分数。<ol>
<li>删除与吸管, 小心不要扰乱颗粒/混浊层, 因为这将污染的分数。吸管可溶性分数为 "可溶性" 标记管。保持冰块。</li>
	</ol>
</li>
	<li>洗涤颗粒与500µL 溶解缓冲 (x2) 和离心机在4°c 在 1.5万 x g 在每洗涤以后5分钟;如果有些多云的层被 pipetted 了, 这是可以的。</li>
	<li>最后清洗后, 取出所有剩余的洗涤缓冲器只留下组织颗粒。 注意: 从这一点看, 不溶性样品现在应该保存在室温下。</li>
	<li>并用重悬颗粒与 X µL 裂解缓冲剂辅以 4% SDS (体积可根据颗粒大小调整; 请参见表 1)。</li>
	<li>油脂实验每个样品三十年代在室温与探针 sonicator (125 瓦特, 20 赫, 脉冲, 振幅 40%, 看见材料目录)。</li>
	<li>煮沸样品 (轧煮) 30 分钟;离心机简要 (十五年代) 在 6000 x g 为不丢失任何样品。</li>
	<li>执行 DC (洗涤剂兼容) 蛋白质化验 (参见材料表) 或劳瑞蛋白质测定以测量蛋白质浓度 (建议开始稀释1/5 样品在各自的溶解缓冲细胞培养和1/10 为组织裂解物)。 注: 由于高洗涤剂浓度, 蛋白质浓度将必须使用 DC 或劳瑞化验。</li>
	<li>整除样品为50µL 批次, 以避免冻融问题。 注意: 通过将两个分数存储在-20 摄氏度以上, 可以在进一步的生化分析之前暂停协议。</li>
</ol>3. 西方污点和量化
<ol><li>按照其他地方报告的西方印迹1,13通过稀释30µg 样本1:1 在加载缓冲区 (1.6x 加载染料, 1x 减少)。 注:30 µg 的蛋白质每分数是足够的, 但可以调整, 以最佳分辨率。3-8% 三乙酸酯聚酰胺凝胶应用于不溶性分数。推荐用于可溶性分数的 4-12% 双三凝胶。</li>
	<li>煮沸样品 (辗煮) 为5分钟离心机简要地 (十五年代) 在 6000 x g 收集样品。</li>
	<li>根据制造商关于减少样品的协议, 通过 SDS 页和西方印迹分析分数。 注: 当转移到0.45 µM 硝化棉膜时, 不溶性分数最佳。0.45 或0.2 µM 硝化纤维素可用于可溶性分数, 取决于蛋白质的大小的兴趣。对高分子量谷蛋白材料进行最佳转移可能需要进行一些优化。</li>
	<li>染色膜与全蛋白染色 (例如, MEMcode, 见材料表), 以可视化的蛋白质加载效率。 注: 西方印迹可以用平均像素强度进行分析。可溶性馏分可归一化为最适合于实验系统的保家蛋白。不溶性分数作为相对蛋白质丰度通过可逆蛋白染色或组蛋白3在分离印迹上的规范化验证。</li>
</ol>

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作者没有什么可透露的。

为了确保一致性和定量的结果, 上述协议需要一些预防措施。首先, mHTT 在两个组分将自发地形成聚集体随着时间的推移在多个冻融循环, 特别是当在高浓度。因此, 冻结蛋白质眼影的整除数是至关重要的, 在按照上文议定书所述的方法运行化验之前, 只解冻所需的体积。此外, 如果不溶性分数产生白色沉淀后解冻, 重建可能是必要的额外的 5 s 超声波脉冲, 5 分钟煮沸, 并重新定量之前通过生化化验分?...

蛋白质质量控制网络的中断, 确保适当折叠和降解细胞蛋白, 可能是阿尔茨海默病 (AD)、帕金森病 (PD)、亨廷顿氏病 (HD) 和其他 "蛋白质错误折叠" 病理学的核心。异常2,3。因此, 对 proteostasis 网络组件及其对病理学的贡献的详细了解, 对于发展改进的治疗干预至关重要。hd 是由 hd 基因内的 CAG 重复异常扩展引起的, 导致杭丁顿蛋白 (HTT) 蛋白4中 polyglutamines (polyQ) 的扩张.HD 发病机制的一个一致的病理特征是产生的错误折叠, 积累和聚集的 HTT 在大脑区域和周围组织, 这已经表明干预的几种方式, 正常细胞稳态和功能5,6. 虽然 HTT 无所不在表达, 纹状体中的中刺神经元有选择性的脆弱性, 最明显的影响与显着皮质萎缩也与 HD 发病机制。
HD 患者和动物模型脑中 HTT 聚集的形成, 通常作为 mHTT7的疾病进展和显性负增益功能的代理标记。虽然蛋白质错误折叠和聚集可能导致 mHTT 毒性的精确机制仍不清楚, 但在 HD 患者和各种动物模型脑中形成这些包裹体是一个不变的、不可避免的特征。夹杂物似乎主要由累积的 HTT 片段组成, 其中包含 N 终端扩展 polyQ, 表明对全长 HTT 的分解和处理以及可选拼接8,9可能会起到在 HD 发病机制中的重要作用. n-端 HTT 片段可能构成一种病理形式的 HTT, 可以快速聚合, 并加速或传播聚合过程10。
然而, 这些包裹体的存在不一定与 HTT 诱导的毒性或细胞死亡有关11。HTT 已被建议从可溶性单体通过可溶性寡聚物种和β薄片纤维到不溶性骨料和夹杂物的聚合过程中, 12.通过标准生化检测方法解决这些不同的蛋白质物种在低洗涤剂裂解缓冲液中的稳定性和使用标准生化检测的难以可视化方面一直是一个挑战。因此, 方法学考虑对于检测累积和聚集 mHTT 的程度和模式至关重要。
这里提出的协议提供了一种方法, 以可视化几个中间体的 HTT, 特别是形成一个不溶性的高分子量 HTT 物种, 似乎密切跟踪 HD 发病机制和疾病进展1,13 ,14。能够解决和跟踪多种 mHTT 为研究人员提供了一种生化工具来研究疾病的发病机制和评估潜在的治疗干预, 通过其调节和对疾病发病机制的影响。

<table>
	<tbody>
		<tr>
			<td>Sterile Filter</td>
			<td>Millipore</td>
			<td>SCGP505RE</td>
			<td>Screw cap, sterile vaccum filter</td>
		</tr>
		<tr>
			<td>1 mL Tissue Grinder, Dounce</td>
			<td>Wheaton</td>
			<td>357538</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Qsonica</td>
			<td>Model Q125</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Protein Assay</td>
			<td>Biorad</td>
			<td>5000111</td>
			<td>Comparable to Lowry assay</td>
		</tr>
		<tr>
			<td>Tris 1M buffer solution</td>
			<td>Alfa Aesar</td>
			<td>J60636</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 5M solution</td>
			<td>Teknova</td>
			<td>S0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS solution</td>
			<td>Teknova</td>
			<td>S0295</td>
			<td></td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Sigma</td>
			<td>E1271</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfony flouride</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>create 100mM stock solution in 100%EtOH, store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td>Create 0.5M stock solution in water</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma</td>
			<td>S4504</td>
			<td>Create 500mM stock solution in water</td>
		</tr>
		<tr>
			<td>Anti-HTT</td>
			<td>Millipore</td>
			<td>MAB5492</td>
			<td>Use 1:1000 for western blot, 1:500 for filter retardation assay</td>
		</tr>
		<tr>
			<td>Anti-GAPDH</td>
			<td>Novus Biologicals</td>
			<td>NB100-56875</td>
			<td>Use 1:1000 for soluble western blot</td>
		</tr>
	</tbody>
</table>

fractionation for resolution of soluble and insoluble huntingtin species

错误折叠蛋白的积累是亨廷顿病 (HD) 和许多其他神经退行性疾病的病理中枢。具体来说, HD 的一个关键病理特征是突变 HTT (mHTT) 蛋白在高分子量复合体中的异常积累, 以及由片段和其他蛋白质组成的细胞内包裹体内。传统的测量和理解各种形式的 mHTT 的贡献的方法包括荧光显微术, 西方印迹分析, 过滤器陷阱化验。
然而, 这些方法大多是构象特异性的, 因此由于聚合溶解度和分辨率的复杂性, 无法解决 mHTT 蛋白通量的全态。
为确定聚合 mHTT 和各种改良的形式和配合物, 细胞团聚体和碎片的分离和增溶是强制性的。在这里, 我们描述了一种分离和可视化可溶性 mHTT、单体、寡聚物、片段和不溶性高分子量 (mHTT) 积累的方法。高分子量蛋白 mHTT 跟踪与疾病进展, 对应于鼠标行为读数, 并已受益于某些治疗干预措施1。这种方法可用于小鼠大脑, 周边组织和细胞培养, 但可以适应其他模型系统或疾病的上下文。

可利用西方分析和过滤器延迟检测方法 (图 2) 测定可溶性和不溶性细胞裂解物后的分解。例如, HEK293T 细胞使用转染试剂 (例如, 脂质体 2000), 与 HTT 外显子1编码 cDNA 含有97谷氨酰胺重复15 其次是聚脯氨酸丰富的区域, 这些细胞被允许用5µM MG132 对44小时的细胞进行了治疗, 以阻止蛋白酶体或亚砜在收获和裂解前的18小时的控制。根...

Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

本文介绍了一种从小鼠脑和细胞培养中分离不溶性和可溶性突变体杭丁顿蛋白的方法。该方法对杭丁顿蛋白蛋白通量和艾滋病在分析疾病发病机制中蛋白质稳态和扰动存在时的定性和定量具有重要意义。

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 ...

fractionation-for-resolution-soluble-insoluble-huntingtin

Research

JoVE Journal

Biochemistry

9.5K 次观看.  University of California Irvine. 本文介绍了一种从小鼠脑和细胞培养中分离不溶性和可溶性突变体杭丁顿蛋白的方法。该方法对杭丁顿蛋白蛋白通量和艾滋病在分析疾病发病机制中蛋白质稳态和扰动存在时的定性和定量具有重要意义。

视频: Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

人死后脑中洗涤剂不溶性蛋白的富集

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

用两个稳定 Isotopically 标记鞘磷脂种鞘磷脂的定量和定性方法

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

利用相扑融合策略生成国产、未加标签的杭丁顿蛋白 Exon1 单体和纤维

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

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 ...

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

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

高速离心条件下 u937 细胞的细胞分裂

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

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 ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

用于活细胞超分辨率显微镜和单分子跟踪的传统 BODIPY 结合物

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...