وقد تم تمويل هذا العمل من قبل المعاهد الوطنية للصحة منح GM086822 ، الأمل معهد القلب جائزة العلوم الأساسية ، وموردوك MJ الخيرية كلية العلوم الاستئماني برنامج البحوث. حدد الكواشف الكهربي وقدمت بسخاء صورة برامج التحليل بواسطة بيو راد.

<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>

حدد الكواشف الكهربي وقدمت صورة برامج التحليل بواسطة بيو راد.

ويمكن تكييف مقايسة المذكورة أعلاه لاختبار عدد لا يحصى من الظروف التي تؤثر على الإجراءات كوصي الآلية كوصي hsp90/hsp70-based فضلا GR ملزمة الستيرويد. بل هو تعديل أساليب ذكرت سابقا 4،8،9،17 ويهدف إلى الاستفادة من التطورات الحديثة في مجال التكنولوجيا الكهربائي ويمكن الوصول ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف </th><th> شركة </th><th> فهرس العدد </th><th> تعليقات </th></tr><tr><td> Sf9 خلايا الحشرات </td><td> Invitrogen </td><td></td><td> للتعبير عن الفيروسة العصوية GR الماوس المؤتلف </td></tr><tr><td> L929 الخلايا </td><td> ATCC </td><td> CRL - 2173 </td><td> لإعداد الماوس الذاتية العصارة الخلوية GR (البديل في التعبير الفيروسة العصوية) </td></tr><tr><td> FiGR خلايا ورم هجين </td><td> ATCC </td><td> CRL - 2173 </td><td> لإعداد استسقاء تحتوي على مكافحة GR ريتوكسيماب (بديل للأجسام المضادة BuGR2 المنقى) </td></tr><tr><td> إكمال المصغر مثبط البروتياز ، EDTA خالية </td><td> روش العلوم التطبيقية </td><td> 11836170001 </td><td></td></tr><tr><td> بروتين A - Sepharose </td><td> سيغما الدريخ </td><td> P3391 </td><td></td></tr><tr><td> الأرنب بالشبكيات lysate </td><td> الأخضر هكتارا (ولاية أوريغون ، ويسكونسن) </td><td></td><td> الغنية مصدر الآلات الذاتية كوصي hsp90/hsp70 </td></tr><tr><td> الكرياتين فسفوكيناز </td><td> سيغما الدريخ </td><td> C3755 </td><td> لتجديد نظام ATP - </td></tr><tr><td> فسفوكرياتين </td><td> سيغما الدريخ </td><td> P7936 </td><td> لتجديد نظام ATP - </td></tr><tr><td> GR المضادة للأجسام المضادة وحيدة النسيلة الماوس (BuGR2) </td><td> بيرس / الحرارية العلمية </td><td> MA1 - 510 </td><td> تستخدم لامتزاز مناعي الموارد الوراثية والضد الأولية كما هو الحال في غرب النشاف </td></tr><tr><td> hsp90 المضادة للأجسام المضادة وحيدة النسيلة الماوس (AC88) </td><td> انزو العلوم الحياتية </td><td> ADI - SPA - 830 </td><td> تستخدم الأجسام المضادة الأولية في غرب النشاف </td></tr><tr><td> hsp70 المضادة للأجسام المضادة وحيدة النسيلة الماوس (N27F3 - 4) </td><td> انزو العلوم الحياتية </td><td> ADI - SPA - 820 </td><td> تستخدم الأجسام المضادة الأولية في غرب النشاف </td></tr><tr><td> مفتش من المصل الماوس </td><td> سيغما الدريخ </td><td> 038K7690 </td><td> كما تستخدم للسيطرة على الضد nonimmune السلبية للامتزاز مناعي </td></tr><tr><td> مكافحة فأر مفتش - البيروكسيداز المتقارن </td><td> سيغما الدريخ </td><td> A4416 </td><td> تستخدم الأجسام المضادة الثانوية في غرب النشاف </td></tr><tr><td> نظام Experion ميكروفلويديك الكهربائي </td><td> الحيوي راد </td><td> 7007001 </td><td> بديل لالتقليدية SDS - PAGE </td></tr><tr><td> [1،2،4،6،7 -- 3 H] ديكساميثازون </td><td> PerkinElmer </td><td> NET1192001MC </td><td> اتباع احتياطات السلامة </td></tr></tbody></table>

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

Hsp90 هو ضروري للغاية وفيرة من البروتين كوصي الجزيئية التي تم العثور على تنظيم أكثر من 150 إشارة حقيقية النواة البروتينات ، بما في ذلك عوامل النسخ (المستقبلات النووية على سبيل المثال ، البروتين p53) وتحركات البروتينات (مثل SRC ، راف ، AKT كيناز) المشاركة في دورة الخلية ، تكون الأورام ، موت الخلايا المبرمج ، وعدة مسارات إشارات حقيقية النواة 1،2. كثير من هذه البروتينات &quot;عميل&quot; لhsp90 ، والتجمع من مستقبلات الستيرويد • hsp90 المجمعات هو أفضل تعريف (الشكل 1). نقدم هنا لتلقي تكيف جلايكورتيكود (اليونان) وفحص مناعي في المختبر GR • أسلوب إعادة hsp90 التي يمكن استخدامها بسهولة لتحقيق حقيقية النواة hsp90 النشاط الوظيفي ، hsp90 بوساطة مستقبلات الستيرويد يجند ملزمة ، ومتطلبات العامل المساعد كوصي الجزيئية. على سبيل المثال ، يمكن استخدام هذا الاختبار لاختبار متطلبات العامل المساعد hsp90 وتأثيرات خارجية مضيفا المركبات لعملية إعادة. كان نظام GR مفيدة بشكل خاص لدراسة hsp90 لأنه يجب أن تكون ملزمة لمستقبلات hsp90 أن يكون لها ملزم يجند فتح المشقوق يمكن الوصول إلى 3 الستيرويد. الذاتية ، GR unliganded موجودة في السيتوبلازم من خلايا الثدييات ملزمة لnoncovalently hsp90. كما وجدت في الموارد الوراثية المحلية • hsp90 heterocomplex ، وملزم يجند GR المشقوق مفتوحة وقادرة على الستيرويد ملزمة. إذا تم استعادة hsp90 تنأى عن GR أو إذا تحول دون وظيفتها ، ومستقبلات غير قادر على ربط الستيرويد ويتطلب إعادة تشكيل لGR • hsp90 heterocomplex قبل النشاط ملزمة الستيرويد 4. يمكن immunoprecipitated GR العصارة الخلوية من خلية باستخدام الأجسام المضادة وحيدة النسيلة ، والبروتينات مثل hsp90 المعقد للGR يمكن يعاير بواسطة لطخة غربية. يمكن تحديد النشاط ملزمة الستيرويد لGR immunoprecipitated التي تفرخ في immunopellet مع [3 H] الستيرويد. وقد أظهرت التجارب السابقة hsp90 بوساطة افتتاح GR ملزم يجند المشقوق يتطلب hsp70 ، وهو second كوصي الجزيئية الأساسية أيضا لبقاء الخلية حقيقية النواة. وحفز النشاط الكيميائي الحيوي لhsp90 hsp70 والبروتينات التي هوب المشترك كوصي ، hsp40 ، و 5 P23. وتعتبر الأجهزة التي تحتوي على كوصي multiprotein hsp90 ، hsp70 ، هوب ، والتطور الطبيعي hsp40 هي موجودة في السيتوبلازم الخلية حقيقية النواة ، وlysate بالشبكيات يوفر كوصي الغنية مصدر بروتين 6. في أسلوب عرضه ، هو immunoadsorbed GR من العصارة الخلوية المحمولة وتجريده من آلات كوصي hsp90/hsp70 الظروف الذاتية باستخدام ملح خفيف. ومن ثم حضنت GR الملح مع تجريده lysate بالشبكيات ، ATP ، و+ K ، والذي ينتج إعادة تشكيل GR • hsp90 heterocomplex وتفعيل النشاط ملزمة الستيرويد 7. ويمكن استخدام هذا الأسلوب لاختبار تأثير العوامل المساعدة كوصي مختلف البروتينات الرواية ، والتجريبية ، أو مثبطات hsp90 GR من أجل تحديد أهميتها الفنية بشأن 8-11 hsp90 بوساطة ملزمة الستيرويد.

Watch this Scientific Journal Video about Biochemical Reconstitution of Steroid Receptorâ€¢Hsp90 Protein  Complexes and Reactivation of Ligand Binding at JoVE.com

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

و<em&gt; في المختبر</emيوصف&gt; طريقة لإعداد مستقبلات جلايكورتيكود الوظيفية (GR) • مجمعات البروتين hsp90 المنقى من البروتينات وlysates الخلوية. أسلوب يستخدم للامتزاز مناعي المؤتلف GR تليها تجريد الملح وإعادة تشكيل البروتين المعقدة. وتناقش أهمية العوامل المساعدة والشروط العازلة ، كما هي تطبيقات أسلوب المحتملة.

إعادة إعماره البيوكيميائية من مستقبلات البروتين الستيرويد • المجمعات Hsp90 وإعادة تنشيط يجند تجليد

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

biochemical-reconstitution-steroid-receptorhsp90-protein-complexes

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

16.3K Views.  Seattle University. و في المختبر طريقة لإعداد مستقبلات جلايكورتيكود الوظيفية (GR) • مجمعات البروتين hsp90 المنقى من البروتينات وlysates الخلوية. أسلوب يستخدم للامتزاز مناعي المؤتلف GR تليها تجريد الملح وإعادة تشكيل البروتين المعقدة. وتناقش أهمية العوامل المساعدة والشروط الع...

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

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

تحديد بروتين المجمعات مع كمية البروتينات في البيرة س.

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

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

تحليل البروتين المجمعات الكبيرة التي الطيف الكتلي الإنشائية

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

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 metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

المعايرة الكيميائية الحيوية من الجليكوجين<em&gt; في المختبر</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

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

البروتين طريقة خالية من تنقية تجليد تحديد الانجذاب من قبل Thermophoresis الميكروسكيل

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

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

تحديد التعديلات بعد متعدية من النباتات بروتين المجمعات

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

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

رسم الخرائط على أساس فؤاد للخصائص مطاطا جدران الخلايا: في الأنسجة، الخلوية، وقرارات التحت خلوية

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

تحديد تفاعلات البروتين يجند عن طريق المسح الضوئي التفاضلية Fluorimetry

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 plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

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

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

في المختبر إعادة تشكيل مجمعات خفيفة حصاد النباتات والطحالب الخضراء

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

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

إعادة إعماره الوظيفية وآخر قناة قياسات تنقية Wildtype والمسخ CFTR البروتين

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

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

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

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.