Name: intracellular refolding assay
Uploaded: 2012-01-24T13:00:00
Description: Watch this Scientific Journal Video about Intracellular Refolding Assay at JoVE.com

达尼洛Maddalo是作为年轻的调查组，从卡尔斯鲁厄理工学院（YIG）的研究奖学金获得者。

1。播种细胞<ol><li>开始前，热身在37℃水浴中培养液，PBS 1X和胰蛋白酶˚彗星</li><li>的孵化器中取出细胞，吸中等。 </li><li>轻轻地适用于洗细胞5毫升的PBS 1X。 </li><li>吸出PBS 1X和适用于1毫升胰蛋白酶含0025％EDTA。 </li><li>轻轻旋转板有均匀分布的胰蛋白酶。 </li><li>在孵化器中放置的细胞在37˚C为5-10分钟（取决于细胞类型）。从不定期检查，如果细胞分离摇板。 </li><li>在10-20毫升培养基重悬细胞，?...

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	<li>Bukau, B., Weissman, J., Horwich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperones+and+protein+quality+control.">Molecular chaperones and protein quality control.</a> Cell. 125, 443-443 (2006).</li><li>Ritossa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+the+heat+shock+response.">Discovery of the heat shock response.</a> Cell Stress Chaperones. 1, 97-97 (1996).</li><li>Hendrick, J. P., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+functions+of+heat-shock+proteins.">Molecular chaperone functions of heat-shock proteins.</a> Annu. Rev. Biochem. 62, 349-349 (1993).</li><li>Fuqua, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+proteins+and+drug+resistance.">Heat shock proteins and drug resistance.</a> Breast. Cancer. Res. Treat. 32, 67-67 (1994).</li><li>Guzhova, I., Margulis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperone+as+a+survival+factor+in+cell+pathology.">Hsp70 chaperone as a survival factor in cell pathology.</a> Int. Rev. Cytol. 254, 101-101 (2006).</li><li>Whitesell, L., Lindquist, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP90+and+the+chaperoning+of+cancer.">HSP90 and the chaperoning of cancer.</a> Nat. Rev. Cancer. 5, 761-761 (2005).</li><li>Konstantinopoulos, P. A., Papavassiliou, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17-AAG:+mechanisms+of+antitumour+activity.">17-AAG: mechanisms of antitumour activity.</a> Expert. Opin. Investig. Drugs. 14, 1471-1471 (2005).</li><li>Galluzzi, L., Giordanetto, F., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+HSP70+for+cancer+therapy.">Targeting HSP70 for cancer therapy.</a> Mol. Cell. 36, 176-176 (2009).</li><li>Ozacmak, V. H., Barut, F., Ozacmak, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+provides+neuroprotection+by+reducing+oxidative+stress+and+HSP70+expression+during+chronic+cerebral+hypoperfusion+in+ovariectomized+rats.">Melatonin provides neuroprotection by reducing oxidative stress and HSP70 expression during chronic cerebral hypoperfusion in ovariectomized rats.</a> J. Pineal. Res. 47, 156-156 (2009).</li><li>Schroder, H., Langer, T., Hartl, F. U., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DnaK,+DnaJ+and+GrpE+form+a+cellular+chaperone+machinery+capable+of+repairing+heat-induced+protein+damage.">DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.</a> EMBO. J. 12, 4137-41 (1993).</li><li>Thulasiraman, V., Matts, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geldanamycin+on+the+kinetics+of+chaperone-mediated+renaturation+of+firefly+luciferase+in+rabbit+reticulocyte+lysate.">Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate.</a> Biochemistry. 35, 13443-13443 (1996).</li><li>Howarth, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp40+molecules+that+target+to+the+ubiquitin-proteasome+system+decrease+inclusion+formation+in+models+of+polyglutamine+disease.">Hsp40 molecules that target to the ubiquitin-proteasome system decrease inclusion formation in models of polyglutamine disease.</a> Mol. Ther. 15, 1110-1110 (2007).</li><li>Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bag1+functions+in+vivo+as+a+negative+regulator+of+Hsp70+chaperone+activity.">Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity.</a> Mol. Cell. Biol. 20, 1083-1083 (2000).</li></ol>

在这项工作中来衡量细胞内分子伴侣复性活动的协议。整个实验可在3到4天，在图概述所示。 1。 萤火虫和海肾荧光素酶产生的光信号的鲁棒性和线性代表一个坚实的基础协议的重现性。 检测的关键步骤是一个有效率的转染试剂的选择，以确保分子伴侣和萤火虫荧光素酶的过度表达。报告基因可交付也与其他方法，如腺病毒?...

在这个实验中使用一个光度计形式Perkin Elmer公司&#39;1420发光反“矢量之光” 方案的光度计： 海肾：100μL注射泵1 反应缓冲液：Pump1，70μL注入荧光素：Pump2，30μl注入对于缓冲区准备在双蒸水，这些股票的解决方案： <ul style="list-style:none"><li> - 1M 硫酸镁 ：246,48克硫酸镁4 * 7H 2 O在1公升</li><li> - 0.5米EGTA：190,18克EGTA 1升</li><li> - 1M KH 2 PO 4：136,09克KH 2 PO 4 1升</li><li> - 1M K2 HPO 4：228,23克K 2 HPO 4 3H 2 O在1公升</li><li> - 5M的 NaCl：292,2 g氯化钠1升</li><li> - 0.5米EDTA：186,12克EDTA · 2H 2 O在1公升&nbsp;</li></ul>甘氨酸 - 甘氨酸缓冲： <ul style="list-style:none"><li> - 25MM Glycylglycin </li><li> - 4硫酸镁15MM </li><li> - 4MM EGTA </li></ul>对于1公升使用3,3克Glycylglycin，一个1M的硫酸镁溶液和一个0.5米EGTA解决方案8毫升15毫升。解决水，调节pH值7,8和带来一个总体积为1升。 海肾/腔肠缓冲区的反应缓冲液： <ul style="list-style:none"><li> - ＆NBSP; 13.4毫米KH 2 PO 4 </li><li> - 86,6毫米KH 2 PO 4 </li><li> - 0,5 M氯化钠</li><li> - 1mm的EDTA </li></ul>对于1升使用13.4毫升一个1M KH 2 PO 4溶液中，一个1M K 2 HPO 4溶液，100毫升一个5M的NaCl溶液和一个0.5米的EDTA溶液2毫升86,6毫升。 <table border="1"><tr><td></td><td> 公司 </td><td> 目录/产品编号： </td></tr><tr><td> DMEM（贝科的修改鹰中等1X） </td><td> GIBCO公司</td><td> 41966029 </td></tr><tr><td> 胎儿牛血清黄金 </td><td>临时机场管理局</td><td> A15 - 151 </td></tr><tr><td> 9基因的X极端 DNA转染试剂</td><td>罗氏公司</td><td&gt; 06365809001 </td></tr><tr><td> PBS 贝科的磷酸盐缓冲盐水1X </td><td> GIBCO公司</td><td> 14190-094 </td></tr><tr><td> MOPS 3 - （N -吗啉）丙磺酸</td><td> Sigma - Aldrich公司</td><td> M1254 </td></tr><tr><td> 放线菌酮 </td><td> Sigma - Aldrich公司</td><td> C7698 </td></tr><tr><td> 被动裂解缓冲5X </td><td> Promega公司</td><td> E194A </td></tr><tr><td> Glycylglycin EGTA 硫酸镁 （硫酸镁4 * 7H 2 O） </td><td> Sigma - Aldrich公司 罗斯 罗斯</td><td> G7278 3054.2 P027.1 </td></tr><tr><td> KH 2 PO 4 K 2 HPO 4（K 2 HPO 4 * 3H 2 O） 氯化钠 EDTA </td><td>罗斯 罗斯 罗斯 罗斯</td><td> 3904.1 6878.2 3957.1 8043.1 </td></tr><tr><td> 荧光素萤火虫 </td><td> Biosynth </td><td> L8200 </td></tr><tr><td> 腔肠 </td><td> Biosynth </td><td> C7000 </td></tr><tr><td> DTT（二硫苏糖醇） </td><td>罗斯</td><td> 6908.2 </td></tr><tr><td> ATP（三磷酸腺苷） </td><td>罗氏公司</td><td> 10519987001 </td></tr></table>

intracellular refolding assay

本协议描述了一个方法来衡量，在基于单元的系统和抑制/刺激活性的化合物可能产生的影响酶的活性分子伴侣。分子伴侣是参与调节蛋白质折叠1的蛋白质，有促进细胞存活时强调侮辱，如热休克2，营养饥饿和接触化学品 /毒药3的关键作用。出于这个原因的伴侣被发现在参与活动，如肿瘤的发展，癌细胞4 chemioresistance以及神经退行性疾病5。能够抑制或刺激这些酶的活性小分子的设计，因此，用于癌症治疗和神经退行性疾病的研究最多的战略之一。这里所描述的实验提供的可能性来衡量一个特定的分子伴侣复性的活动，并研究对比赛的影响ounds就其活动。这种方法调查的分子伴侣基因转染的萤火虫荧光素酶基因的表达载体。它已经描述，可以通过10,11分子伴侣复性变性萤火虫荧光素酶。作为转染控制正常化，为海肾荧光素酶基因的载体转染。在本协议中所述的所有转染基因的X极端 11（罗氏）在HEK - 293细胞。在第一步中，蛋白质的合成，抑制细胞治疗与放线菌酮。此后蛋白展开是在45 ° C诱导热休克30分钟。恢复后，在37 ° C，蛋白质重新折叠成其活性构象和萤火虫荧光素酶的活性是用作读出：会产生更多的光，更多的蛋白质将重新获得原来的构象。非热震惊细胞作为参考（100％的复性荧光素酶）。

Watch this Scientific Journal Video about Intracellular Refolding Assay at JoVE.com

Intracellular Refolding Assay

在这个协议的方法来衡量热休克后细胞内蛋白质复性的描述。这种方法可以用来研究如分子伴侣和他们的共同因素或化合物能够影响他们的活动foldases。萤火虫荧光素酶的活动是作为记者来衡量伴侣复性活动。

细胞内复性检测

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Research

JoVE Journal

Biology

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

成像APP的细胞内运输与光活化GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

纯化及复性的淀粉样纤维（他）<sub&gt; 6</sub&gt; -tagged重组Shadoo蛋白表达的包涵体<em&gt;大肠杆菌</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...