Name: in situ subcellular fractionation of adherent and non-adherent mammalian cells
Uploaded: 2010-07-23T17:30:00
Description: Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

Anyaporn Sawasdichai Nazefah阿卜杜勒哈米德感谢泰王国政府和马来西亚政府，分别为博士奖学金。这项工作是由威康信托项目荣获PSJ和KG授予。我们也感谢布里斯托尔大学欧胜生物成像设施。

一，准备进行分馏本节介绍了聚- L -赖氨酸涂层显微镜的盖玻片和细胞附着之前分馏的准备。如果需要的细胞可以瞬时转染与蛋白表达载体之前或之后附件。 A.制备聚- L -赖氨酸涂布盖玻片<ol><li>准备一个1mg/ml的聚- L -赖氨酸的蒸馏水溶液。 </li><li>清洁盖玻片大衣与聚- L -赖氨酸孵化摇椅平台上至少1小时的解决方案，在22 ° C。 </li><li>用无菌蒸馏水冲洗涂?...

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	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

共同的问题和建议： 在清洗过程中的全部或大部分细胞都将丢失。在洗涤步骤中的液体必须避免盖玻片的6孔​​板边缓慢加入。同样的液体，应仔细倾斜板中删除，并慢慢地移液多余的液体。使用聚- L -赖氨酸涂布盖玻片，可提高细胞粘附。 基因组DNA是没有完全消化。对于某些类型的细胞的DNA酶I消化步骤，可能需要进行扩展和/或DNA酶I浓度增加，以达到彻...

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<td>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
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<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
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<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
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<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>EGTA</td>
<td>Chemical</td>
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<td>E4378-25G </td>
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<td>Chemical</td>
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<td>P-6757</td>
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<td>S/8600/53</td>
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<td>Triton X-100</td>
<td>Chemical</td>
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<td>T-6876</td>
<td>&nbsp;</td>
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<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
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<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
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<td>Histone H1</td>
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<td>Santa Cruz</td>
<td>SC-8030</td>
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<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
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<td>Tubulin-&alpha;</td>
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<td>Santa Cruz</td>
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in situ subcellular fractionation of adherent and non-adherent mammalian cells

蛋白质的功能是紧密耦合的蛋白定位。虽然一些蛋白质被限制到特定的位置或亚细胞车厢，许多蛋白质是自由扩散，作为一个自由交流的的一个亚群，是与特定的亚细胞域或结构紧密相关的人口。允许的可视化，在原地的亚细胞分馏蛋白质的条块分割，还可以揭示蛋白质亚群，定位于特定的结构。例如，去除可溶性的细胞质蛋白和松散举行的核蛋白质可以揭示一些与染色质的转​​录因子的稳定协会。随后的DNA消化，可以揭示在某些情况下，协会与网络，统称为核脚手架或核基质蛋白和RNA的。 在这里，我们描述了在显微镜盖玻片壁和非贴壁的哺乳动物细胞原位分馏过程中所需的步骤。使用特异性抗体或荧光融合蛋白和荧光显微镜，可以达到蛋白质的可视化。抗体和/或行为，作为特定的车厢或结构的标记的荧光染料允许蛋白定位进行详细的映射。原位分馏也可以结合免疫印迹比较每个分数中蛋白质的数额。这个简单的生化方法可以揭示协会，否则仍未被发现。

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

允许在原位显微镜盖玻片上的哺乳动物细胞的亚细胞分馏蛋白定位可视化。

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Research

JoVE Journal

Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

科研

生物学

MISSION esiRNA for RNAi Screening in Mammalian Cells

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Clonogenic Assay: Adherent Cells

克隆形成含量：贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Analysis of Cell Cycle Position in Mammalian Cells

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...