Name: utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes
Uploaded: 2020-11-09T15:45:00
Description: Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

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。...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+analysis+revelas+stable+multiprotein+complexes+in+both+fission+and+budding+yeasts+containing+Myb-related+Cdc5p/Cef1p,+novel+pre-mRNA+splicing+factors,+and+snRNAs.">Proteomics analysis revelas stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.</a> Molecular and Cellular Biology. 22 (7), 2011-2024 (2002).</li><li>Lourenco, R. F., Leme, A. F. P., Oliveira, C. 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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 <e...

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20...

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。

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

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