Name: detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans
Uploaded: 2018-07-30T13:00:00
Description: Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

本研究使用的C. 线虫菌株取自秀丽遗传学中心, 由国立卫生研究院 (P40 OD010440) 提供支持。这项工作得到了 NIH 的支持: 1R03AG048578-01, 纽约市立大学-CCRG 1501 至俊良, 和 PSC-纽约市立大学 66184-00 44 和 67248-00 45 至俊良。我们感谢凯西野蛮-邓恩亲切地分享她的实验室空间。所有荧光图像都收集在皇后学院的核心设施。我们感谢威廉 j. 赖斯对手稿的评论。

1. 解决方案
<ol><li>
NGM 板
	<ol>
<li>在2升锥形烧瓶中, 加入3克氯化钠、17克琼脂、2.5 克蛋白胨和975毫升的 dH2o 盖上铝箔的烧瓶口。将烧瓶蒸压50分钟, 冷却20–30分钟。</li>
		<li>然后添加灭菌的解决方案: 1 毫升的1米 CaCl2, 1 毫升5毫克/毫升胆固醇在乙醇, 1 毫升1米 MgSO4, 25 毫升1米 KPO4 (pH 6.0) 缓冲。漩涡的解决方案, 以混合良好。</li>
		<li>使用液体分配器, 将 ...

<ol>
	<li>Essers, M. A., 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=FOXO+transcription+factor+activation+by+oxidative+stress+mediated+by+the+small+GTPase+Ral+and+JNK.">FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK.</a> The EMBO Journal. 23 (24), 4802-4812 (2004).</li><li>Oh, S. W., 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=JNK+regulates+lifespan+in+Caenorhabditis+elegans+by+modulating+nuclear+translocation+of+forkhead+transcription+factor/DAF-16.">JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (12), 4494-4499 (2005).</li><li>Gross, A., 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=Caspase+cleaved+BID+targets+mitochondria+and+is+required+for+cytochrome+c+release,+while+BCL-XL+prevents+this+release+but+not+tumor+necrosis+factor-R1/Fas+death.">Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death.</a> The Journal of Biological Chemistry. 274 (2), 1156-1163 (1999).</li><li>Doerflinger, M., Glab, J. A., Puthalakath, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BH3-only+proteins:+a+20-year+stock-take.">BH3-only proteins: a 20-year stock-take.</a> The FEBS Journal. 282 (6), 1006-1016 (2015).</li><li>Mayorova, T. 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=Cells+containing+aragonite+crystals+mediate+responses+to+gravity+in+Trichoplax+adhaerens+(Placozoa),+an+animal+lacking+neurons+and+synapses.">Cells containing aragonite crystals mediate responses to gravity in Trichoplax adhaerens (Placozoa), an animal lacking neurons and synapses.</a> PLoS One. 13 (1), e0190905 (2018).</li><li>Terron-Camero, L. C., Molina-Moya, E., Sanz-Fernandez, M., Sandalio, L. M., Romero-Puertas, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+and+nitrogen+species+(ROS/RNS)+during+hypersensitive+cell+death.">Detection of reactive oxygen and nitrogen species (ROS/RNS) during hypersensitive cell death.</a> Methods in Molecular Biology. , 97-105 (2018).</li><li>Pedrazzini, E., Vitale, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biosynthesis+and+maturation+in+the+ER.">Protein biosynthesis and maturation in the ER.</a> Methods in Molecular Biology. , 179-189 (2018).</li><li>Liang, J., Shaulov, Y., Savage-Dunn, C., Boissinot, S., Hoque, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+intracellular+channel+proteins+respond+to+heat+stress+in+Caenorhabditis+elegans.">Chloride intracellular channel proteins respond to heat stress in Caenorhabditis elegans.</a> PLoS One. 12 (9), e0184308 (2017).</li><li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+and+microinjection.">Transformation and microinjection.</a> Wormbook. , (2006).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods in Cell Biology. 48, 451-482 (1995).</li><li>Yochem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nomarski+images+for+learning+the+anatomy,+with+tips+for+mosaic+analysis.">Nomarski images for learning the anatomy, with tips for mosaic analysis.</a> Wormbook. , (2006).</li><li>Dulhunty, A., Gage, P., Curtis, S., Chelvanayagam, G., Board, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glutathione+transferase+structural+family+includes+a+nuclear+chloride+channel+and+a+ryanodine+receptor+calcium+release+channel+modulator.">The glutathione transferase structural family includes a nuclear chloride channel and a ryanodine receptor calcium release channel modulator.</a> The Journal of Biological Chemistry. 276 (5), 3319-3323 (2001).</li><li>Suh, K. S., Malik, M., Shukla, A., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4,+skin+homeostasis+and+cutaneous+cancer:+surprising+connections.">CLIC4, skin homeostasis and cutaneous cancer: surprising connections.</a> Molecular Carcinogenesis. 46 (8), 599-604 (2007).</li><li>Yao, Q., Qu, X., Yang, Q., Wei, M., Kong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4+mediates+TGF-beta1-induced+fibroblast-to-myofibroblast+transdifferentiation+in+ovarian+cancer.">CLIC4 mediates TGF-beta1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer.</a> Oncology Reports. 22 (3), 541-548 (2009).</li><li>Ronnov-Jessen, L., Villadsen, R., Edwards, J. C., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+a+chloride+intracellular+channel+gene,+CLIC4,+in+transforming+growth+factor-beta1-mediated+conversion+of+fibroblasts+to+myofibroblasts.">Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1-mediated conversion of fibroblasts to myofibroblasts.</a> The American Journal of Pathology. 161 (2), 471-480 (2002).</li><li>He, G., 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=Role+of+CLIC4+in+the+host+innate+responses+to+bacterial+lipopolysaccharide.">Role of CLIC4 in the host innate responses to bacterial lipopolysaccharide.</a> European Journal of Immunology. 41 (5), 1221-1230 (2011).</li><li>Zhong, J., 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=Inhibition+of+CLIC4+enhances+autophagy+and+triggers+mitochondrial+and+ER+stress-induced+apoptosis+in+human+glioma+U251+cells+under+starvation.">Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation.</a> PLoS One. 7 (6), e39378 (2012).</li><li>Liang, J., 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=The+Caenorhabditis+elegans+schnurri+homolog+sma-9+mediates+stage-+and+cell+type-specific+responses+to+DBL-1+BMP-related+signaling.">The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling.</a> Development. 130 (26), 6453-6464 (2003).</li><li>Leopold, L. E., Heestand, B. N., Seong, S., Shtessel, L., Ahmed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+pairing+during+meiosis+triggers+multigenerational+transgene+silencing+in+Caenorhabditis+elegans.">Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (20), E2667-E2676 (2015).</li><li>Schultz, R. D., Bennett, E. E., Ellis, E. A., Gumienny, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+extracellular+matrix+organization+by+BMP+signaling+in+Caenorhabditis+elegans.">Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.</a> PLoS One. 9 (7), e101929 (2014).</li><li>Matus, D. Q., 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=Invasive+cell+fate+requires+G1+cell-cycle+arrest+and+histone+deacetylase-mediated+changes+in+gene+expression.">Invasive cell fate requires G1 cell-cycle arrest and histone deacetylase-mediated changes in gene expression.</a> Developmental Cell. 35 (2), 162-174 (2015).</li><li>Reddy, K. C., Villeneuve, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+HIM-17+links+chromatin+modification+and+competence+for+initiation+of+meiotic+recombination.">C. elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination.</a> Cell. 118 (4), 439-452 (2004).</li><li>Ames, K., 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=A+non-cell-autonomous+role+of+BEC-1/BECN1/Beclin1+in+coordinating+cell-cycle+progression+and+stem+cell+proliferation+during+germline+development.">A non-cell-autonomous role of BEC-1/BECN1/Beclin1 in coordinating cell-cycle progression and stem cell proliferation during germline development.</a> Current Biology. 27 (6), 905-913 (2017).</li><li>Pekar, O., 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=Linking+the+environment,+DAF-7/TGFbeta+signaling+and+LAG-2/DSL+ligand+expression+in+the+germline+stem+cell+niche.">Linking the environment, DAF-7/TGFbeta signaling and LAG-2/DSL ligand expression in the germline stem cell niche.</a> Development. 144 (16), 2896-2906 (2017).</li><li>Schvarzstein, M., 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=DNA+helicase+HIM-6/BLM+both+promotes+MutSgamma-dependent+crossovers+and+antagonizes+MutSgamma-independent+interhomolog+associations+during+caenorhabditis+elegans+meiosis.">DNA helicase HIM-6/BLM both promotes MutSgamma-dependent crossovers and antagonizes MutSgamma-independent interhomolog associations during caenorhabditis elegans meiosis.</a> Genetics. 198 (1), 193-207 (2014).</li><li>Flemming, A. J., Shen, Z. Z., Cunha, A., Emmons, S. W., Leroi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+polyploidization+and+cellular+proliferation+drive+body+size+evolution+in+nematodes.">Somatic polyploidization and cellular proliferation drive body size evolution in nematodes.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (10), 5285-5290 (2000).</li><li>Nagamatsu, Y., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+the+control+of+body+size+by+a+G-kinase+and+a+downstream+TGFbeta+signal+pathway+in+Caenorhabditis+elegans.">Mechanisms for the control of body size by a G-kinase and a downstream TGFbeta signal pathway in Caenorhabditis elegans.</a> Genes to Cells. 9 (1), 39-47 (2004).</li></ol>

本文提出了一种快速有效的方法来验证蛋白质亚细胞变化从细胞质到细胞核。蛋白表达由 GFP 融合显示, 而核结构由 DAPI 染色验证 (图 3)。由于染色线虫蛋白具有挑战性, 大多数C. 线虫蛋白亚细胞定位的特点是标记它们与标记蛋白, 如 GFP, 拉扎, mCherry, 和其他18,19,20,21. ...

蛋白质亚细胞定位的变化是反应热应激、饥饿、氧化应激、细胞凋亡、蛋白质磷酸化等内部或外部信号的一种常见机制。例如, 热应力诱导一个 foxo 蛋白成员 DAF-16 核易位1,2和亲凋亡 BCL-2 蛋白出价 translocates 的线粒体后, 接受死亡信号3,4。可以使用各种技术来检测这些更改。西方印迹和生化分离亚细胞结构 (如线粒体或细胞核) 的组合可以很好地实现目标3。然而, 它需要一个特定的抗体对抗蛋白质的兴趣。因此, 一个完善的抗体成为成功的关键。另一种方法是标签不同的亚细胞结构或细胞器与各种标记, 如绿色荧光蛋白 (GFP), 红色荧光蛋白 (RFP), 黄色荧光蛋白 (YFP), 和 mCherry, 同时标签的蛋白质与其他标记的兴趣。然后, 观察他们在共聚焦显微镜下, 以本地化目标5,6。放射性同位素是标记靶蛋白, 然后检测其亚细胞定位7的替代选择。但是, 这种方法需要对放射性废料进行适当的训练和处理。在诸如缺乏特定抗体、缺少适当的标记或 scarceness 等设备的情况下, 需要考虑一种替代方法。为了确定蛋白质核易位, 它是有吸引力的仅标记目标蛋白质与标记和染色核与化学试剂 4 ', 6-diamidino-2-苯基吲哚 (DAPI), 因为这只需要常规荧光显微镜。
Immunolabeling线虫具有抗体是挑战, 由于低渗透性的蛋壳或胶原角质层周围的动物。同时, 由于线虫蛋白与脊椎动物的 orthologs 有明显的差异, 一些商业公司提供特定产品的c. 线虫。小实验室很难为自己生成线虫抗体。社区的研究人员经常使用标记蛋白作为标记来显示蛋白质定位或基因表达。本文以 EXL-1::GFP 为例, 在热应力作用下跟踪蛋白质核易位8。将一个集成的平移exl-1::gfp到动物基因组中, 用于稳定地表达gfp融合的基因。研究表明, exl-1在肠道、体壁肌和其他亚细胞结构中表达8。该协议演示如何将蠕虫同步到第四幼虫 (L4) 阶段, 进行热应激实验, 并在常规荧光显微镜下进行 DAPI 染色、乙醇固定、丙酮固定和成像。

<table>
	<tbody>
		<tr>
			<td>DAPI</td>
			<td>Biotium</td>
			<td>40043</td>
			<td>both Hoechst 33258 and Hoechst 33342 also works well</td>
		</tr>
		<tr>
			<td>OP50</td>
			<td>CGC</td>
			<td>OP50</td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Kim Wipe</td>
			<td>Kimberly-Clark Corporation</td>
			<td></td>
			<td>soft tissue</td>
		</tr>
		<tr>
			<td>Zeiss&#160; AX10</td>
			<td>Zeiss</td>
			<td></td>
			<td>fluorescence microscope</td>
		</tr>
		<tr>
			<td>Axiovision Rel 4.8</td>
			<td>Zeiss</td>
			<td></td>
			<td>microscope software</td>
		</tr>
		<tr>
			<td>AxioCam MR Rev3</td>
			<td>Zeiss</td>
			<td></td>
			<td>digital camera</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent nail polish gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-20 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-200 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum wire worm pick</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans

本协议采用绿色荧光蛋白 (GFP) 融合蛋白和 4 '、6-diamidino-2-苯基吲哚 (DAPI) 染色法跟踪蛋白质亚细胞定位变化;特别是在热应力条件下的核易位。蛋白质的反应与外部和内部信号相应。一种常见的机制是改变其亚细胞定位。本文介绍了一种跟踪蛋白质定位的协议, 不需要抗体, 放射性标签, 或共焦显微镜。在这篇文章中, GFP 是用来标记的目标蛋白 EXL-1 在C. 线虫, 一个成员的氯离子胞内通道蛋白 (CLICs) 家族, 包括哺乳动物 CLIC4。采用转化与γ辐射相结合的法, 建立了一种集成的转化exl-1::gfp转基因线 (具有启动子和全基因序列), 并稳定地表达了该基因和gfp。最近的研究表明, 在热应力, 而不是氧化应激, EXL-1::GFP 积累在细胞核。将 GFP 信号与细胞核结构和 DAPI 信号重叠, 证实了 EXL-1 亚细胞在应力作用下的定位变化。本协议提出两种不同的固定方法 DAPI 染色: 乙醇固定和丙酮固定。本文提出的 DAPI 染色协议具有快速、高效的功效, 同时保留了 GFP 信号和蛋白质亚细胞定位的变化。此方法只需要使用 Nomarski、FITC 过滤器和 DAPI 过滤器的荧光显微镜。适用于小型实验室设置、本科生研究、高中学生研究、生物技术教室。

氯离子胞内通道蛋白 (点击) 是一种在12种高度保守的多功能蛋白质。许多研究表明, CLICs 调节细胞的压力, 自噬, 凋亡, 癌变, 血管生成和巨噬细胞先天免疫反应在哺乳动物系统13,14,15,16 ,17。线虫中有两个点击同系物: exl-1...

Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

该协议演示了如何在热应激下跟踪蛋白质核易位, 方法是使用绿色荧光蛋白 (GFP) 融合蛋白作为标记, 4 ', 6-diamidino-2-苯基吲哚 (DAPI) 染色。DAPI 染色协议快速, 保留了 GFP 和蛋白质亚细胞定位信号。

秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Research

JoVE Journal

Biology

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

MBP基于标记策略的Mgm101重组蛋白的制备

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

高尔基蛋白质荧光质量中心的定量定位

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...