Name: desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells
Uploaded: 2018-04-25T15:00:00
Description: Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

这项工作得到了瑞士国家科学基金会 Sinergia 赠款 CRSII3 147697, 基金会《应用 Krebsforschung 和苏黎世 Krebsliga 的支持。

1. 胞浆和核蛋白分数的制备
<ol><li>板 4 x 106 ACC-MESO-4 细胞在 T150 细胞培养瓶中生长 RPMI 1640 培养基补充10% 血清, 1x 青霉素/链霉素 (100x), 2 毫米 l-谷氨酰胺 (200 毫米)。当细胞达到 80-90% (~ 5-5. 5 x 106细胞) 的汇合处时, 继续进行蛋白质提取核和胞浆分数。 注: ACC-MESO-4 细胞线是从山本理 BioResource 中心11获得的。</li>
	<li>吸入培养基, 通过加入15毫升的 1x PBS 来洗涤细胞,...

<ol>
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L., Yong, J., Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+and+post-transcriptional+gene+regulation.">RNA-binding proteins and post-transcriptional gene regulation.</a> FEBS Letters. 582 (14), 1977-1986 (2008).</li><li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+biological+roles+of+alternative+3'UTRs.">Evolution and biological roles of alternative 3'UTRs.</a> Trends in Cell Biology. 26 (3), 227-237 (2016).</li><li>Matoulkova, E., Michalova, E., Vojtesek, B., Hrstka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+3'+untranslated+region+in+post-transcriptional+regulation+of+protein+expression+in+mammalian+cells.">The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells.</a> RNA Biology. 9 (5), 563-576 (2012).</li><li>von Roretz, C., Di Marco, S., Mazroui, R., Gallouzi, I. 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3 ' UTRs 属于非编码基因组3, 所有非编码 rna 可以与蛋白质相互作用, 以发挥其功能7。当哺乳动物基因组被发现被普遍转录并产生长非编码 rna 16 的很大一部分时, 新的证据表明这些长非编码 rna 在调节基因表达时的作用与染色质重塑复合体17。这种知识是利用 RNA 下拉法得到的。因此, 要开始破译特定 RNA 的 interactors, 第一步可能?...

基因表达和基因产物的最终水平可以通过影响 mrna 稳定性和 mrna 翻译率1来严格调节。这些转录后调控机制是通过非编码 rna 和/或 rna 结合蛋白 (限制性商业惯例) 与靶向 mRNA 的相互作用而施加的。它通常是 3 ' 未翻译的 mRNA 区域 (3 ' UTR-属于基因组2) 的非编码部分, 其中包含特定的cis-管理元素 (), 由反演作用因素 (如 miRNA 或限制性商业惯例) 识别。3. 最好研究的cis元素在 3 ' UTR, 是腺嘌呤丰富的元素 (是) 的主题, 这是公认的特定的 AU 结合蛋白 (AUBP), 反过来, 诱导 mRNA 退化/deadenylation (是介导的衰变) 或mRNA 稳定化4。
3 ' UTR 的 calretinin mRNA (CALB2) 的大小是 573 bp 长, 包含一个假定的 AUUUA pentamer, 如 AREsite2 的预测, 生物信息学工具 5.与假定的存在相一致的是主题, pmirGLO 向量-报告分析显示了这个元素在 CALB2 mRNA6中的稳定作用。最后, 用体外RNA 下拉法确定了通过 AUBP 稳定 calretinin mRNA 的方法。
由于所有非编码 rna 与蛋白质7相互作用,体外rna 下拉是确定 rna interactors 以帮助破译分子机制的一种很好的方法和首选的检测手段。在这个 rna 下拉法, 商业上被合成的 rna 探针, 被标记以被改进的生物素 (desthiobiotin) 的形式在 RNA 链的 3 ' 末端被使用了。rna desthiobiotin 是固定的通过与链亲和素的相互作用的磁性珠子, 这是用来拉下来的蛋白质, 特别是与绑定 RNA 的利益。非变性和活性蛋白从间皮瘤细胞胞浆的比例被用作蛋白质的来源。这种 RNA 束缚的蛋白质是洗脱从磁性珠子, 运行通过 12% SDS 页凝胶, 转移到膜, 并探讨了不同的抗体。
在标准的链亲和素-生物素亲和纯化过程中, 需要苛刻的变性条件来破坏强不可逆转的生物素-链亲和素键洗脱绑定蛋白 8, 这可能导致离解蛋白质复合物。与生物素不同的是, desthiobiotin 可逆地结合到链亲和素, 并以缓冲的生物素溶液进行竞争性置换, 允许蛋白质的温和洗脱, 避免自然生物素化分子的分离 9,表明该技术是理想的分离本地蛋白质复合体在当地条件下。
体外约束条件和严格性是由盐浓度、还原剂和洗涤剂百分比决定的, 应与细胞上下文中存在的情况相近, 以便识别真正的体内交互作用。在此之前所实现的绑定条件已被证明是适当的, 以确定虚宾作为一种金结合蛋白10。这种方法可以节省时间, 因为优化适当的绑定条件可能会耗费时间和挑战性。此外, 该方法可作为任何 RNA 下拉实验的启动协议, 通过改变盐和洗涤剂的浓度, 改变甘油的百分比, 添加其他盐, 可以逐步优化。此外, 我们证明, 即使是一个短的25核苷酸 RNA 探针窝藏 pentamer 是-主题可以用来证明与特定 AUBP 的互动。

<table border="0" cellpadding="0" cellspacing="0" width="1340">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">NE-PER Nuclear and cytoplasmic extraction kit</td>
			<td style="width:295px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:124px;">78833</td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Protease Inhibitor Tablets, EDTA-free</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:124px;">A32965</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce Magnetic RNA-protein Pull-Down Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20164</td>
			<td>Includes magnetic beads and therefore the kit need to be stored at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pierce RNA 3&#8217; End Desthiobiotinylation Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20163</td>
			<td style="width:468px;">The kit is a part of the &#34;Pierce Magnetic RNA-protein Pull-Down Kit&#34;, and needs to be stored at -20 &#176;C unlike the pull-down kit (4 &#176;C).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2,&#160; magnetic stand</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">12321D</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:453px;">Anti - HuR (MOUSE) monoclonal antibody</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">-</td>
			<td style="width:468px;">The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit&#160; - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - &#945; - tubulin (MOUSE) monoclonal antibody</td>
			<td>Santa Cruz</td>
			<td style="width:124px;">8035</td>
			<td>1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - PARP (RABBIT) Polyclonal antibody</td>
			<td>Cell Signaling</td>
			<td>9542</td>
			<td>1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - Mesothelin (MOUSE) Monoclonal Antibody&#160;</td>
			<td>Rockland</td>
			<td>200-301-A87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary goat anti-rabbit antibody</td>
			<td style="width:295px;">Cell Signaling</td>
			<td style="width:124px;">7074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary rabbit anti-mouse antibody</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A-9044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RPMI - 1640 medium</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FBS - Filtrated Bovine Serum</td>
			<td style="width:295px;">Pan Biotech&#160;</td>
			<td style="width:124px;">P40-37500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Penicillin - streptomycin (100x)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">L-Glutamine solution (200 mM)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">0.25% Trypsin-EDTA (1x)</td>
			<td style="width:295px;">Gibco by Life technologies&#160;</td>
			<td style="width:124px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN 3 Cell</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini Protean System Glass Plates&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 &#181;L</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra Cell Casting Modules</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1658050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNaseZap RNase Decontamination Solution</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:453px;">Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1</td>
			<td style="width:295px;">Roth</td>
			<td style="width:124px;">3029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">20% SDS&#160;</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td>A3942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">PVDF transfer membrane&#160;</td>
			<td style="width:295px;">Perkin Elmer</td>
			<td style="width:124px;">NEF1002</td>
			<td>Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Trans-Blot SD semi-dry transfer cell&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td>1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Clarity Western ECL Substrate</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1705061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FusionFX&#160; Digital Imager</td>
			<td style="width:295px;">Vilber</td>
			<td style="width:124px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNA oligo synthesis</td>
			<td style="width:295px;">Mycrosynth AG, Switzerland</td>
			<td style="width:124px;">-</td>
			<td>the synthesis scale - 0.04 &#181;mol - HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Bovine serum albumin</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Hydrochloric Acid (HCl) 6 M</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td style="width:124px;">182883.1211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ultra Pure 1 M Tris, pH 7.5&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">15567027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Glycerol&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td>17904</td>
			<td>50% sterile aliquots to be stored at -20&#176;C</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:453px;">Pierce Streptavidin magnetic beads&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">88817</td>
			<td style="width:468px;">Please note that these magnetic beads have an average diameter of 1 &#181;m (range from 0.5 - 1.5 &#181;m). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 &#181;m, are also used in RNA-pulldown but be aware that this may affect purification process.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">TEMED</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ammonium persulfate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Coomassie G-250</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A3480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminiumsulfate-(14-18)-hydrate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">368458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ortho-phosphoric acid&#160;</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A0637</td>
			<td></td>
		</tr>
	</tbody>
</table>

desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells

体外RNA 下拉仍然主要用于确定 rna 结合蛋白识别特定 rna 结构和图案的协议的第一步。在这个 rna 下拉协议, 商业上合成的 rna 探针被标记以修改的形式生物素, desthiobiotin 在 RNA 链的 3 ' 末端, 可逆地束缚到链亲和素并且因而允许蛋白质洗脱在更加生理上条件。rna desthiobiotin 是固定的, 通过与链亲和素的相互作用, 磁性珠子, 这是用来拉下来的蛋白质, 特别是与 RNA 的利益。非变性和活性蛋白从间皮瘤细胞胞浆的比例被用作蛋白质的来源。此处描述的方法可用于检测已知的 rna 结合蛋白与含有一系列兴趣序列的25核苷酸 (nt) 长 RNA 探针之间的相互作用。这对于完成使用报告向量法实现的 RNA 分子中稳定或不稳定元素的功能表征是有用的。

在这个实验中, 一个 25 nt 长片段的 calretinin 3 ' UTR 窝藏是主题 (CALB2 3 ' UTR (是) 25 nt) 被用来测试它是否结合了人类抗原 R (虚) 蛋白, 一个已知的 mRNA 稳定剂。为了测试这些元素的特异性, 25-nt RNA 探针 CALB2 3 ' UTR (mtARE) 包含了一个有主题的突变, 以前被证明是为了取消 "是" 的主题的稳定效果, 使用了6。第三个 RNA 探针代表负控制, 这是一个 28 nt 无关的 rna, 它包...

Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin-链亲和素亲和介导纯化间皮瘤细胞 RNA 相互作用蛋白的研究

Desthiobiotin 标记的合成 25-核苷酸 RNA 寡核苷酸, 其中含有腺嘌呤丰富的元素 (是) 的主题, 允许特定的结合胞浆是结合蛋白。

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

Research

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Cancer Research

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Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

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The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

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Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

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Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...

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Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

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