Эта работа финансировалась Национальным институтом здоровья грант GM086822, Надежда института сердца фундаментальных наук награду, и MJ Мердок Благотворительный фонд колледжа науки исследовательской программы. Выберите электрофореза реагенты и программное обеспечение для анализа изображений щедро предоставляемые Bio-Rad.

1. Подготовка цитозоле клеток, содержащих функциональные GR <ol><li> Техническое примечание: Все буферы должны храниться в холодильнике, и каждый шаг этого протокола, в том числе центрифугирования и инкубации, должны быть выполнены на льду или при температуре 4 ° С, если не указано иное. Низ?...

<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>

Анализа, описанного выше, может быть адаптирована для проверки множества условий, влияющих на действия шаперонов hsp90/hsp70-based техники шаперона а также GR стероидных обязательными. Она является модификацией ранее сообщалось методы 4,8,9,17 и предназначен, чтобы воспользоваться последн?...

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th><th> Комментарии </th></tr><tr><td> Sf9 клетках насекомых </td><td> Invitrogen </td><td></td><td> бакуловирус для экспрессии рекомбинантных GR мыши </td></tr><tr><td> Клеток L929 </td><td> АТСС </td><td> CRL-2173 </td><td> для эндогенных мыши GR цитозоле подготовки (альтернатива бакуловирус выражение) </td></tr><tr><td> FiGR клетки гибридомы </td><td> АТСС </td><td> CRL-2173 </td><td> для подготовки асцит содержащие анти-GR моноклональных антител (альтернатива очищенной BuGR2 антитела) </td></tr><tr><td> Полное-Mini ингибитор протеазы, ЭДТА без </td><td> Roche Applied Science </td><td> 11836170001 </td><td></td></tr><tr><td> белок-сефарозой </td><td> Sigma-Aldrich </td><td> P3391 </td><td></td></tr><tr><td> ретикулоцитов кролика лизат </td><td> Зеленый Га (штат Орегон, WI) </td><td></td><td> богатым источником эндогенной hsp90/hsp70 техники шаперона </td></tr><tr><td> креатинфосфокиназы </td><td> Sigma-Aldrich </td><td> C3755 </td><td> для АТФ-система регенерации </td></tr><tr><td> фосфокреатина </td><td> Sigma-Aldrich </td><td> P7936 </td><td> для АТФ-система регенерации </td></tr><tr><td> анти-GR мышиных моноклональных антител (BuGR2) </td><td> Пирс / Thermo Scientific </td><td> MA1-510 </td><td> используется для GR иммуноадсорбции и как первичных антител в западных промокательной </td></tr><tr><td> анти-Hsp90 мышиных моноклональных антител (AC88) </td><td> Энцо наук о жизни </td><td> ADI-SPA-830 </td><td> используются в качестве первичных антител в западных промокательной </td></tr><tr><td> анти-hsp70 мышиных моноклональных антител (N27F3-4) </td><td> Энцо наук о жизни </td><td> ADI-SPA-820 </td><td> используются в качестве первичных антител в западных промокательной </td></tr><tr><td> IgG в сыворотке мыши </td><td> Sigma-Aldrich </td><td> 038K7690 </td><td> используется в качестве неиммунной антител для отрицательного контроля иммуноадсорбции </td></tr><tr><td> анти-мышиных IgG-пероксидазы сопряженным </td><td> Sigma-Aldrich </td><td> A4416 </td><td> использованы в качестве вторичных антител в западных промокательной </td></tr><tr><td> Experion микрожидкостных электрофореза системы </td><td> Bio-Rad </td><td> 7007001 </td><td> альтернативой традиционным SDS-PAGE </td></tr><tr><td> [1,2,4,6,7 - 3 H] дексаметазон </td><td> PerkinElmer </td><td> NET1192001MC </td><td> следовать технике безопасности </td></tr></tbody></table>

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

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

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

<em&gt; В пробирке</em&gt; Способ получения функциональных глюкокортикоидных рецепторов (ГР) • Hsp90 белковых комплексов из очищенных белков и клеточных лизатов описано. Метод использует иммуноадсорбции рекомбинантных GR следует соли зачистки и восстановления белкового комплекса. Важность кофакторов и буферных условия обсуждаются, равно как и потенциальные области применения метода.

Биохимические Восстановление стероидных рецепторов • Hsp90 белковые комплексы и Реактивация связывания лигандов

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

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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

Идентификация белковых комплексов с количественной протеомики в CEREVISIAE С.

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

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Анализ Большие белковые комплексы структурных масс-спектрометрии

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

Биохимический Титрование гликогена<em&gt; В пробирке</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

Очистки белков-Free метод привязки Определение Affinity по Микромасштабные Термофорез

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

Идентификация пост-трансляционных модификаций растительных белковых комплексов

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

Определение белков-лиганд с использованием дифференциальной сканирующей флуориметрии

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

Экстракорпоральное возобновлении деятельности светособирающих комплексов растений и зеленых водорослей

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

Функциональная Реорганизация и активности канала измерения очищенной дикого типа и мутантных CFTR белка

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

Определение на основе экономического роста и биохимическая Подтверждение генетическим требованиям белка деградации в<em&gt; Saccharomyces Cerevisiae</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

Генетические и биохимические подходы к<em&gt; В Vivo</em&gt; и<em&gt; In Vitro</em&gt; Оценка белка олигомеризации: The Рианодин Рецептор Case Study

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