这项工作得到了FAPESP赠款（15/06477-9）的支持。

1. 酵母总提取物制备
<ol><li>用适当的氨基酸或核基，将酵母细胞与TAP标签9 融合成1 L YNB-胶质（酵母氮基补充2%m/v葡萄糖）的拼接因子之一， 在这种情况下，腺苷（20 μg/mL）、柳辛（30微克/mL）、色氨酸（30微克/mL），最高可达OD600 = 1.0。</li>
	<li>在 4 °C 下以 17，000 x g 的 10 分钟在 3 个 500 mL 离心机瓶中离心，从 1 L 培养物中收集细胞，然后用 10 mL 冷无菌水洗两次。</li>
	<li>将收集的酵母细胞重新用于冷缓冲器 A 的 1/10 细胞体积 （10 mmol L-1 HEPES pH = 7.9， 1.5 mmol L-1 MgCl2， 50 mmol L-1 KCl， 5% v/v 甘油， 0.5 mmol L-1 DTT， EDTA 无蛋白酶抑制剂鸡尾酒）.。</li>
	<li>将小滴细胞悬浮液氮冷冻。小滴可以通过管道重用电池溶液和直接将 50 μL 滴入液氮中获得。 警告:液氮可能会在与皮肤或眼睛接触时造成伤害。使用拨配的安全设备进行处理。 注:协议可以在此处暂停。电池悬浮液的冻结滴可以存储在 -80 °C 下。</li>
	<li>准备酵母细胞解解，在球磨机设备中研磨，以20 Hz/s为6个周期，持续3分钟。 注:每个周期后，将容器浸入液氮中，以避免熔化。协议可以在此处暂停。冷冻提取物可储存在 -80 °C 下。 酵母拼接提取物也可以使用同质化物解解球体10，或使用迫击炮和害虫11，12。</li>
	<li>将含有这些提取物的管子在室温下放入水中，偶尔摇晃，从而融化提取物。</li>
	<li>离心机提取物在 45，000 x g 1 h 在 4 °C.</li>
	<li>通过BCA方法13量化清除超高纳特的蛋白质含量。</li>
	<li>准备提取物的无名小品，快速将其冷冻在液氮中，并储存在 -80 °C 下。 注:协议可以在此处暂停。</li>
</ol>2. 甘油梯度制备
<ol><li>在缓冲区 A 中准备两个甘油溶液，一个包含 10% v/v，另一个包含 30% v/v 甘油。</li>
	<li>在 30% v/v 甘油溶液中将交叉链接剂谷胱甘肽添加到 0.1% v/v，并混合以实现均质化。 注意事项:使用适当的安全设备处理烟罩内的谷胱甘肽。</li>
	<li>在 12 mL 离心机管（14 x 89 mm）底部加入 6 mL 的冷 10% v/v 甘油溶液。</li>
	<li>加入6 mL的冷30%v/v甘油溶液，辅以谷胱甘肽，将注射器连接到长针上，在管底提供梯度主设备，就在10%v/v甘油溶液下面。 注意:另外，10% v/v 甘油溶液可以在 30% v/v 甘油/谷胱甘肽溶液的顶部小心地管道。</li>
	<li>使用梯度主设备生成连续密度梯度。 注:在梯度主设备中，管子被放置在一个适当的机架中，并根据制造商关于确定参数（时间/角度/速度）的建议进行短暂旋转。在这项工作中，我们使用: 2:25 分钟/81.5°/11 rpm。</li>
	<li>在添加细胞提取物之前，小心地在顶部添加 200 μL 的 7% v/v 甘油/缓冲垫。 注:垫子的甘油浓度必须低于用于创建线性梯度的浓度较低的甘油溶液。</li>
</ol>3. 提取离心机
<ol><li>将大约 2 毫克的总蛋白质加载到每个 12 mL 线性甘油梯度 10%至 30% 谷胱甘肽的顶部，并配有垫子。</li>
	<li>将管子放在预冷却的回转桶转子中。 注意:轻轻处理管子，以避免在超中心处理前混合。</li>
	<li>离心机在 194，000 x g 为 16 h 在 4 °C.</li>
	<li>Aliquot 每个 12 mL 管在 24 500 μL 分数使用适应的 Econosystem 或小心管道。 注:经过改造的 EconoSystem 由渗透泵、紫外线探测器和连接到管穿孔装置的分数收集器组成。沉降剖面可以通过测量280纳米的吸收度来监测。</li>
	<li>使用 40% v/v 甘油溶液通过渗透泵将甘油 10%+30% 梯度从管子推至分数收集器。 注:协议可以在此处暂停。将分数存储在 -80 °C 下，直到使用。</li>
	<li>将每个分数的 50 μL 直接加载到点斑或槽斑点上的硝基纤维素膜上。使用抗体对CBP（1:6，000，检测TAP标签的CBP部分）和与马萝卜过氧化酶（1:15，000）结合的抗兔IgG作为二级抗体，通过免疫膨胀检测蛋白质。</li>
</ol>

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作者没有利益冲突。

蛋白质蛋白和核糖核酸-蛋白质相互作用可以通过交联剂稳定下来。由此产生的复合物必须稳定，能够承受甘油梯度的超中心化。此外，缓冲条件应允许交互，但必须足够严格，以避免非特定的绑定。在下面显示的实验中，我们使用了已经为体外拼接反应15建立的缓冲液。
应针对分析的复合物优化离心的速度和时间。我们测试了不同的沉积条件，在 94，000 x g 下离心取样，在 4°C 下以 16 x g 16h 离心取样，结果相似。这些控件对于确定所分析的复合物不沉淀的条件非常重要。
我们还测试了不同的污点方法，直接在膜上管道，使用槽斑或点斑系统，这表明点斑给出了更可重复的结果。
使用谷胱甘肽作为交联器的一个主要限制是，十字链路无法恢复。因此，蛋白质不能在SDS-PAGE之后通过免疫膨胀进行分析，而只能通过原生凝胶或点斑进行分析。然而，可逆的交联剂，如甲醛，也可能被使用。
我们以前使用蛋白质的共免疫沉淀来识别拼接复合物，但在瞬态或弱相互作用的情况下，交行可能有助于中间复合物的分离，以便以后通过质谱法确定其成分。虽然我们在这里的重点是拼接体，这种方法当然可以用来隔离其他动态过程，包括具有不同成分的中间复合物。

拼接是一个高度动态的过程，需要以协调的方式约束和释放多种因素。这些拼接因子包括RNA结合蛋白、ATPases、螺旋酶、蛋白激酶和磷酸盐、无用性韧带等1、2、3:为了让分子重新排列发生，其中一些因素与拼接体子复杂性非常短暂地结合，使得这些RNP中间复合物的隔离和识别极具挑战性。
在这里， 我们使用Grafix方法4，5来稳定酵母拼接因子Cwc24与B行为复合物6的相互作用，以便识别与该子复合物相关的其他因素，并确定是否 泛素韧带Prp19在Cwc24的结合或释放到19（NTC）复合物和激活前内电的5'端和第一次转录反应中起任何作用地方。使大分子沿着甘油梯度使大分子接触到交叉链接器日益集中的优点是它避免了复合物交叉链路4、5，从而避免了聚合物的形成。
这种方法被用作蛋白质同位素沉淀和拉下测定的补充，尽管允许对大型复合物进行隔离，但对于在大型动态复合物7、8中维持瞬态相互作用可能并不可靠。在甘油梯度中使用固定试剂可以稳定这些因素的结合，从而确认特定蛋白质与拼接亚复合物的相互作用。由于所选的交联器在化学上不可逆转，因此在梯度离心后，通过点斑对恢复分数中的蛋白质进行分析。

<table>
	<tbody>
		<tr>
			<td>Anti-Calmodulin Binding Protein Epitope</td>
			<td>Millipore</td>
			<td>07-482</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-Rabbit IgG</td>
			<td>GE Healthcare</td>
			<td>NA934</td>
			<td></td>
		</tr>
		<tr>
			<td>EconoSystem</td>
			<td>Bio-Rad</td>
			<td>1-800-424-6723</td>
			<td>Parts of the EconoSystem used: peristaltic pump, the UV detector and the fraction collector</td>
		</tr>
		<tr>
			<td>EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Recovery System</td>
			<td>Beckman Coulter</td>
			<td>270-331580</td>
			<td>Tube-perforating device that was connected to the parts of the EconoSystem</td>
		</tr>
		<tr>
			<td>Gradient Master Model 107ip</td>
			<td>Biocomp</td>
			<td>107-201M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer Mill MM 200</td>
			<td>Retsch</td>
			<td>207460001</td>
			<td>Ball Mill device</td>
		</tr>
		<tr>
			<td>Rotor F12-6x500Lex</td>
			<td>Thermo Scientific</td>
			<td>096-062375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall RC 6 Plus Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>36-101-0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging Bucket Rotor P40ST</td>
			<td>Hitachi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge CP 80 NX</td>
			<td>Hitachi</td>
			<td>901069</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear Centrifuge Tubes (14 x 89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes

mRNA 前拼接是一个非常动态的过程，在组装、RNA 处理和复杂组件的释放过程中涉及许多拼接体子复合物的分子重新排列。甘油梯度离心已用于分离蛋白质或RNP（核蛋白）复合物，用于功能和结构研究。在这里，我们描述了Grafix（梯度固定）的利用，这是首次开发用于净化和稳定单粒子低温电子显微镜的大分子复合物，以识别暂时与拼接体复合物结合的拼接因子之间的相互作用。此方法基于将样品离心到固定试剂的浓度增加，以稳定复合物。在甘油梯度上装载酵母总提取物离心后，通过点斑分析回收的分数，以识别拼接体子复合物并确定单个拼接因子的存在。

为了分析 Cwc24-TAP 的沉积剖面，并确定 Grafix 方法是否有效稳定其与拼接亚复杂物的结合，我们通过甘油梯度的离心分离了表达 Cwc24-TAP 的细胞的总酵母提取物，无论是否存在谷胱甘肽作为交联剂。然后，通过带抗体对 TAP 标签的 CBP 部分进行带抗体的插槽斑点分析 24 个 500 μL 分数的样本。结果显示，在没有交联器的情况下，Cwc24集中在分数5和13之间（图1B），相对应于约80至200 MDA14的复合物。然而，在交叉链接器的存在下，Cwc24 在梯度上的位置被转移到梯度底部的分数，与较大的复合物相对应（图 1）。这些结果表明，谷胱甘肽稳定了Cwc24与拼接复合物的关联。
为了确定保留 Cwc24 的复合体是否与由 snRNP U2、U5 和 U6 以及 NTC 复合物形成的 Bact 复合物相对应，我们将 Cwc24 沉积与 U5 snRNP 子单位 Prp8 和 NTC 子单位 Prp19 的沉积进行了比较。 酵母菌株表示这些蛋白质中任何一种融合到 TAP 标签上， 都受到 Grafix 的处理， 其次是点斑，用于检测蛋白质。这三个拼接子单位显示了类似的轮廓，在梯度底部沉积了较大的复合物。Cwc24 集中在相同的分数中，但由于它与拼接体短暂关联，它也存在于较轻的分数中（图 2）。点的量化显示这些配置文件（图2B）。
有趣的是，分数 11 和 12 是 Prp8 和 Prp19 开始集中的部分，这表明这是 Bact 复合物开始沉积的梯度部分。为了确定这些蛋白质与这些分数中的复合物有关，11和12分的白化物在原生凝胶上受到电泳，随后又受到西方斑点的影响。Prp8和Prp19的信号显示为几乎不能进入8%丙烯酰胺凝胶的涂片，表明它们确实是大型复合物的一部分（图3）。Cwc24 也存在于第 12 部分， 但浓度要低得多， 与其与 Bact 复合物的瞬时结合一致。这些结果表明，谷胱甘肽可以用作交联器，以稳定瞬态因素与拼接亚复合物的结合。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61994/61994fig01.jpg"> 图1:格拉菲克斯持有与较大复合物相关的Cwc24。表达Cwc24-TAP的细胞总酵母提取物通过甘油梯度的离心分离，无论是在没有或存在十字链球菌谷胱醛。（A） 梯度的奇数部分因Cwc24-TAP的免疫检测而出现槽斑，该斑点与TAP标签CBP部分的抗体有关。（B） 使用图像 J 对 Cwc24-TAP 进行量化，通过甘油梯度的分数显示蛋白质的浓度。表示是否存在谷胱甘肽。Y 轴通过梯度显示 Cwc24 的相对量，计算为分数中信号的强度，相对于第一个分数（Fn/F1）。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61994/61994fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61994/61994fig02.jpg"> 图2:通过甘油/谷胱甘肽梯度沉降拼接因子。表达 Cwc24、Prp8 或与 TAP 标签融合的酵母细胞的提取物被加载在含有谷胱甘肽的甘油梯度上，并离心用于分离拼接复合物。（A） 梯度的二十四个部分的样本通过点斑和TAP融合蛋白的免疫检测与TAP标签的CBP部分抗体进行分析。（B） 使用图像 J 对蛋白质进行量化，显示其浓度在甘油/谷胱甘肽梯度的底部部分。Y轴通过梯度显示蛋白质的相对量，计算为分数中信号的强度，相对于显示背景上方信号（Fn/F1）的第一个分数。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61994/61994fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61994/61994fig03.jpg"> 图3:通过原生PAGE和免疫布洛特检测拼接复合物。图2中显示的梯度中第11和12个部分的Aliquot通过8%丙烯酰胺原生凝胶上的电泳分离，并受到具有CBP抗体的免疫膨胀。蛋白质没有检测到带，因为复合物太大，无法分离在原生凝胶上。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61994/61994fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

在这里，我们描述了Grafix（梯度固定）的利用，这是一种在交行器面前的甘油梯度离心，用于识别暂时与拼接体复合物结合的拼接因子之间的相互作用。

格拉菲克斯用于检测 糖精 的瞬态相互作用器

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

utilization-grafix-for-detection-transient-interactors-saccharomyces

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

3.5K 次观看.  University of Sao Paulo. 在这里，我们描述了Grafix（梯度固定）的利用，这是一种在交行器面前的甘油梯度离心，用于识别暂时与拼接体复合物结合的拼接因子之间的相互作用。

视频: Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

个性化肽阵列在器官移植 HLA 抗体检测中的研究

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

科研

生物化学

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

通过对Rho激酶活性的暂时抑制,增强人类诱导多能干细胞衍生心肌细胞的诱导

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

贝克酵母糖精中线粒体基因组表达产品的标记和可视化

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

通过代谢标记和薄层色谱分析 糖浆 中中性脂质合成

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

格拉菲克斯用于检测 糖精 的瞬态相互作用器

格拉菲克斯用于检测糖精的瞬态相互作用器