这项工作是由国家健康授予GM086822，希望心脏研究所的基础科学奖，兆焦耳默多克慈善信托基金科学学院研究计划研究院资助。选择电泳试剂和图像分析软件Bio - Rad公司提供的慷慨。

1。制备细胞细胞质中含有功能的GR <ol><li>技术说明：所有的缓冲区应冷藏，此协议，包括离心和孵化，每一步应在冰上或在4 ° C，除非另有说明。低温是必不可少的，以防止GR和蛋白质复合物的降解。 </li><li>使用冷冻离心机，取得了〜1-5毫升颗粒细胞功能的GR表达了高浓度。在遗传资源丰富的细胞来源的例子包括：小鼠成纤维L929细胞，它表达的内源性遗传资源的高浓度，并昆虫重组GR杆状病毒?...

<ol>
	<li>Pratt, W. B., Toft, D. O. <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+signaling+protein+function+and+trafficking+by+the+hsp90/hsp70-based+chaperone+machinery.">Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery.</a> Exp. Biol. Med. 228, 111-133 (2003).</li><li>Murphy, P. J. M. <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+glucocorticoid+receptor+steroid+binding+and+trafficking+by+the+hsp90/hsp70-based+chaperone+machinery:+implications+for+clinical+intervention.">Regulation of glucocorticoid receptor steroid binding and trafficking by the hsp90/hsp70-based chaperone machinery: implications for clinical intervention.</a> Leukemia. 19, 710-712 (2005).</li><li>Pratt, W. B., Morishima, Y., Osawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hsp90+chaperone+machinery+regulates+signaling+by+modulating+ligand+binding+clefts.">The Hsp90 chaperone machinery regulates signaling by modulating ligand binding clefts.</a> J. Biol. Chem. 283, 22885-22889 (2008).</li><li>Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+steroid+receptor&#149;hsp90+heterocomplex+assembly+system+of+rabbit+reticulocyte+lysate.">Reconstitution of the steroid receptor&#149;hsp90 heterocomplex assembly system of rabbit reticulocyte lysate.</a> J. Biol. Chem. 271, 12833-12839 (1996).</li><li>Morishima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hsp+organizer+protein+hop+enhances+the+rate+of+but+is+not+essential+for+glucocorticoid+receptor+folding+by+the+multiprotein+Hsp90-based+chaperone+system.">The Hsp organizer protein hop enhances the rate of but is not essential for glucocorticoid receptor folding by the multiprotein Hsp90-based chaperone system.</a> J. Biol. Chem. 275, 6894-6900 (2000).</li><li>Murphy, P. J. M., Kanelakis, K. C., Galigniana, M. D., Morishima, Y., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry,+abundance,+and+functional+significance+of+the+hsp90/hsp70-based+multiprotein+chaperone+machinery+in+reticulocyte+lysate.">Stoichiometry, abundance, and functional significance of the hsp90/hsp70-based multiprotein chaperone machinery in reticulocyte lysate.</a> J. Biol. Chem. 276, 30092-30098 (2001).</li><li>Dittmar, K. D., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+the+glucocorticoid+receptor+by+the+reconstituted+Hsp90-based+chaperone+machinery.+The+initial+hsp90.p60.hsp70-dependent+step+is+sufficient+for+creating+the+steroid+binding+conformation.">Folding of the glucocorticoid receptor by the reconstituted Hsp90-based chaperone machinery. The initial hsp90.p60.hsp70-dependent step is sufficient for creating the steroid binding conformation.</a> J. Biol. Chem. 272, 13047-13054 (1997).</li><li>Kanelakis, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+the+hsp70-binding+protein+BAG-1+on+glucocorticoid+receptor+folding+by+the+hsp90-based+chaperone+machinery.">Differential effects of the hsp70-binding protein BAG-1 on glucocorticoid receptor folding by the hsp90-based chaperone machinery.</a> J. Biol. Chem. 274, 34134-34140 (1999).</li><li>Morishima, Y., Kanelakis, K. C., Murphy, P. J. M., Shewach, D. S., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+iterative+ratcheting+of+receptor-bound+hsp70+between+its+ATP+and+ADP+conformations+during+assembly+of+glucocorticoid+receptor.hsp90+heterocomplexes.">Evidence for iterative ratcheting of receptor-bound hsp70 between its ATP and ADP conformations during assembly of glucocorticoid receptor.hsp90 heterocomplexes.</a> Biochemistry. 40, 1109-1116 (2001).</li><li>Murphy, P. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pifithrin-alpha+inhibits+p53+signaling+after+interaction+of+the+tumor+suppressor+protein+with+hsp90+and+its+nuclear+translocation.">Pifithrin-alpha inhibits p53 signaling after interaction of the tumor suppressor protein with hsp90 and its nuclear translocation.</a> J. Biol. Chem. 279, 30195-30201 (2004).</li><li>Murphy, P. J. M., Morishima, Y., Kovacs, J. J., Yao, T. P., Pratt, W. B. <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+the+dynamics+of+hsp90+action+on+the+glucocorticoid+receptor+by+acetylation/deacetylation+of+the+chaperone.">Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone.</a> J. Biol. Chem. 280, 33792-33799 (2005).</li><li>Morishima, Y., Murphy, P. J. M., Li, D. P., Sanchez, E. R., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+assembly+of+a+glucocorticoid+receptor&#149;hsp90+heterocomplex+resolves+two+sequential+ATP-dependent+events+involving+first+hsp70+and+then+hsp90+in+opening+of+the+steroid+binding+pocket.">Stepwise assembly of a glucocorticoid receptor&#149;hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket.</a> J. Biol. Chem. 275, 18054-18060 (2000).</li><li>Pratt, W. B., Toft, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+receptor+interactions+with+heat+shock+protein+and+immunophilin+chaperones.">Steroid receptor interactions with heat shock protein and immunophilin chaperones.</a> Endocr. Rev. 18, 306-360 (1997).</li><li>Bodwell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+phosphorylated+sites+in+the+mouse+glucocorticoid+receptor.">Identification of phosphorylated sites in the mouse glucocorticoid receptor.</a> J. Biol. Chem. 266, 7549-7555 (1991).</li><li>Penna, A., Cahalan, M. W. e. s. t. e. r. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blotting+using+the+Invitrogen+NuPage+Novex+Bis+Tris+minigels.">Blotting using the Invitrogen NuPage Novex Bis Tris minigels.</a> J. Vis. Exp. (7), e264-e264 (2007).</li><li>Choo, Y. S., Zhang, Z. <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+Protein+Ubiquitination.">Detection of Protein Ubiquitination.</a> J. Vis. Exp. (30), e1293-e1293 (2009).</li><li>Murphy, P. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+mechanism+of+assembly+of+a+glucocorticoid+receptor&amp;bull;Hsp70+complex+that+is+primed+for+subsequent+Hsp90-dependent+opening+of+the+steroid+binding+cleft.">Visualization and mechanism of assembly of a glucocorticoid receptor&amp;bull;Hsp70 complex that is primed for subsequent Hsp90-dependent opening of the steroid binding cleft.</a> J. Biol. Chem. 278, 34764-34773 (2003).</li><li>Schrum, 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=High-sensitivity+detection+and+quantitative+analysis+of+native+protein-protein+interactions+and+multiprotein+complexes+by+flow+cytometry.">High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.</a> Sci STKE. 389, pl2-pl2 (2007).</li><li>Davis, T. R., Schrum, 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=IP-FCM:+Immunoprecipitation+Detected+by+Flow+Cytometry.">IP-FCM: Immunoprecipitation Detected by Flow Cytometry.</a> J. Vis. Exp. (46), e2066-e2066 (2010).</li><li>Felts, S. J., Karnitz, L. M., Toft, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functioning+of+the+Hsp90+machine+in+chaperoning+checkpoint+kinase+I+(Chk1)+and+the+progesterone+receptor+(PR).">Functioning of the Hsp90 machine in chaperoning checkpoint kinase I (Chk1) and the progesterone receptor (PR).</a> Cell Stress Chaperones. 12, 353-363 (2007).</li><li>Cintron, N. S., Toft, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+requirements+for+Hsp40+and+Hsp70+in+the+Hsp90+chaperone+pathway.">Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway.</a> J. Biol. Chem. 281, 26235-26244 (2006).</li></ol>

上文所述的检测可以适应测试无数影响hsp90/hsp70-based伴​​侣机械伴侣行动以及GR类固醇具有约束力的条件。这是以前报道的方法 4,8,9,17的修改，旨在充分利用电泳技术的最新进展，并接触到更广泛的研究界。可以添加额外的辅助因子或蛋白质的利益，在GR•HSP90 heterocomplex重组的混合物，在本议定书第4步中所述，以观察对类固醇的约束力，heterocomplex蛋白质相互作用，并共同因素的要求影?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> Sf9细胞</td><td> Invitrogen公司</td><td></td><td>杆状病毒表达的重组小鼠GR </td></tr><tr><td> L929细胞</td><td> ATCC </td><td> CRL - 2173 </td><td>内源性鼠标GR细胞质准备（替代杆状病毒表达） </td></tr><tr><td> FiGR杂交瘤细胞细胞</td><td> ATCC </td><td> CRL - 2173 </td><td>准备含有反GR单克隆抗体的腹水（替代纯化BuGR2抗体） </td></tr><tr><td>完整的迷你蛋白酶抑制剂，EDTA无</td><td>罗氏应用科学部</td><td> 11836170001 </td><td></td></tr><tr><td>蛋白A琼脂糖</td><td> Sigma - Aldrich公司</td><td> P3391 </td><td></td></tr><tr><td>兔网织红细胞裂解液</td><td>绿色公顷（俄勒冈州，威斯康星州） </td><td></td><td>内源性hsp90/hsp70伴侣机械的丰富来源</td></tr><tr><td>肌酸磷酸激酶</td><td> Sigma - Aldrich公司</td><td> C3755 </td><td> ATP再生系统</td></tr><tr><td>磷酸肌酸</td><td> Sigma - Aldrich公司</td><td> P7936 </td><td> ATP再生系统</td></tr><tr><td>反GR鼠单克隆抗体（BuGR2） </td><td>皮尔斯/ Thermo Scientific的</td><td> MA1 - 510 </td><td>用于遗传免疫吸附作为第一抗体免疫印迹</td></tr><tr><td>抗- HSP90鼠单克隆抗体（AC88） </td><td>恩佐生命科学</td><td> ADI公司的SPA - 830 </td><td>作为主要抗体用于免疫印迹</td></tr><tr><td>抗HSP70鼠单克隆抗体（N27F3 4） </td><td>恩佐生命科学</td><td> ADI公司的SPA - 820 </td><td>作为主要抗体用于免疫印迹</td></tr><tr><td>从小鼠血清IgG抗体</td><td> Sigma - Aldrich公司</td><td> 038K7690 </td><td>作为非免疫抗体用于免疫吸附阴性对照</td></tr><tr><td>抗鼠IgG -过氧化物酶的共轭</td><td> Sigma - Aldrich公司</td><td> A4416 </td><td>作为第二抗体用于免疫印迹</td></tr><tr><td>的Experion微流体电泳系统</td><td> Bio - Rad公司</td><td> 7007001 </td><td>替代传统的SDS - PAGE </td></tr><tr><td> [1,2,4,6,7 - 3H]地塞米松</td><td>珀金埃尔默</td><td> NET1192001MC </td><td>按照安全防范措施</td></tr></tbody></table>

biochemical reconstitution of steroid receptor&#x2022;hsp90 protein  complexes and reactivation of ligand binding

HSP90是一个重要和非常丰富，已发现调节超过150个的真核细胞信号蛋白，包括转录因子（如核受体，P53），并参与细胞周期蛋白激酶（如SRC，英国皇家空军，Akt激酶）的分子伴侣蛋白，肿瘤的发生，细胞凋亡和多种真核细胞信号转导通路1,2。许多这些“客户”HSP90蛋白，类固醇受体大会•HSP90复合物是最好的定义（ 图1）。我们在座的适应性强的糖皮质激素受体 ​​（GR）的免疫测定和体外GR•HSP90重组的方法，可随时用来探测HSP90真核细胞的功能活动，HSP90介导的类固醇激素受体 ​​配体的结合，以及分子伴侣辅助因子的要求。例如，此法可用于测试HSP90的辅助因子的要求和加入外源性化合物的重组进程的影响。 遗传资源已经为研究HSP90一个特别有用的系统，必须绑定到HSP90，因为受体有一个开放的配体结合裂访问类固醇 3 。内源性的，unliganded的GR非共价的约束，以HSP90的哺乳动物细胞的细胞质中。如发现内源性GR•HSP90 heterocomplex，GR配体结合裂，是开放的和有约束力的类固醇的。如果从GR HSP90不赞成或如果其功能受到抑制，受体是无法绑定类固醇和需要重组的GR•HSP90 heterocomplex的结合活性类固醇之前恢复 4 。遗传资源，可以从细胞细胞质使用单克隆抗体免疫沉淀，如HSP90蛋白复合物，以遗传资源，可以通过免疫印迹检测。免疫沉淀的GR结合活性的类固醇，可以由孵化与[3H]类固醇immunopellet 。 以前的实验已经证明HSP90介导的GR配体裂约束力的开放需要HSP70，第二个分子伴侣，也为真核细胞的活力至关重要。 HSP90和HSP70的生化活性催化合作伴侣蛋白合，hsp40，和P23 5 。一个多蛋白的伴侣机械HSP90，HSP70，合，和hsp40是在真核细胞的细胞质内源性目前，网织红细胞裂解液提供了一个伴侣，含有丰富的蛋白质来源6。 提出的方法，GR是immunoadsorbed从细胞细胞质和内源性hsp90/hsp70伴侣用温和的盐条件下的机械剥离。盐剥离的GR然后孵育•HSP90 heterocomplex和类固醇的结合活性的激活7，网织红细胞裂解液，三磷酸腺苷，和K +，这在重组的GR结果。可以利用这种方法来测试各种伴侣的辅助因子，新的蛋白质，和实验HSP90或GR抑制剂的影响，以确定其HSP90介导的类固醇具有约束力8-11的功能意义。

Watch this Scientific Journal Video about Biochemical Reconstitution of Steroid ReceptorÃ¢â‚¬Â¢Hsp90 Protein  Complexes and Reactivation of Ligand Binding at JoVE.com

Biochemical Reconstitution of Steroid ReceptorÃƒÂ¢Ã¢â€šÂ¬Ã‚Â¢Hsp90 Protein  Complexes and Reactivation of Ligand Binding

一个<em&gt;在体外</em&gt;准备从纯化的蛋白质和细胞裂解液的功能性糖皮质激素受体（GR）•HSP90蛋白复合物的方法描述。该方法利用免疫吸附的盐剥离和蛋白质复合体重组重组的GR。辅助因子和缓冲条件的重要性进行了讨论，作为潜在的方法应用。

类固醇受体的生化重建•Hsp90的蛋白质复合物和配体激活绑定

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Research

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Biology

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Identification of protein complexes with quantitative proteomics in S. cerevisiae

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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生物学

Analyzing Large Protein Complexes by Structural Mass Spectrometry

大蛋白复合物结构质谱分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Biochemical Titration of Glycogen In vitro

Biochemical Titration of Glycogen In vitro

糖原生化滴定<em&gt;在体外</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Identification of Post-translational Modifications of Plant Protein Complexes

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

在植物和绿藻的捕光配合体外重组

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

成长型测定和遗传需求生化确认的蛋白质降解<em&gt;酿酒酵母</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

遗传和生物化学方法<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;蛋白齐聚评估：兰尼碱受体案例研究

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...