ونحن نشكر الدكتور تاكيشي إيواتسوبو (جامعة طوكيو، اليابان) يرجى تقديم والبلازميدات ترميز WT 3xFLAG-LRRK2، وطفرات. كما نشكر الدكتور داريو اليسي (جامعة داندي، المملكة المتحدة) لتوفير يرجى MLi-2 وبلازميد ترميز Rab10 هكتار. وأيد هذا العمل "الجمعية اليابانية" لتعزيز العلوم (JSPS) كاكينهي المنحة رقم JP17K08265 (غي).

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J. 473, 2671-2685 (2016).</li><li>Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K., Koike, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphate-binding+tag,+a+new+tool+to+visualize+phosphorylated+proteins.">Phosphate-binding tag, a new tool to visualize phosphorylated proteins.</a> Mol. Cell. Proteomics. 5 (4), 749-757 (2006).</li><li>Using a Hemacytometer to Count Cells. J. Vis. Exp Available from: <a target="_blank" href="https://jove.unicartagenaproxy.elogim.com/science-education/5048/using-a-hemacytometer-to-count-cells">https://jove.unicartagenaproxy.elogim.com/science-education/5048/using-a-hemacytometer-to-count-cells</a> (2017)</li><li>Ni, D., Xu, P., Gallagher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+Immunodetection.">Immunoblotting and Immunodetection.</a> Curr. Protoc. Mol. Biol. (114), 10.8.1-10.8.37 (2016).</li><li>Reith, A. 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=GSK2578215A;+a+potent+and+highly+selective+2-arylmethyloxy-5-substitutent-N-arylbenzamide+LRRK2+kinase+inhibitor.">GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor.</a> Bioorg. Med. Chem. Lett. 22 (17), 5625-5629 (2012).</li><li>Fell, M. 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=MLi-2,+a+potent,+selective+and+centrally+active+compound+for+exploring+the+therapeutic+potential+and+safety+of+LRRK2+kinase+inhibition.">MLi-2, a potent, selective and centrally active compound for exploring the therapeutic potential and safety of LRRK2 kinase inhibition.</a> J. Pharmacol. Exp. Ther. 355, 397-409 (2015).</li><li>Dzamko, N., 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+LRRK2+kinase+activity+leads+to+dephosphorylation+of+Ser(910)/Ser(935),+disruption+of+14-3-3+binding+and+altered+cytoplasmic+localization.">Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization.</a> Biochem. J. 430 (3), 405-413 (2010).</li><li>Th&#233;venet, J., Pescini Gobert, R., Hooft van Huijsduijnen , R., Wiessner, C., Sagot, Y. J. <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+LRRK2+expression+points+to+a+functional+role+in+human+monocyte+maturation.">Regulation of LRRK2 expression points to a functional role in human monocyte maturation.</a> PLoS One. 6 (6), e21519 (2011).</li></ol>

الكتاب ليس لها علاقة بالكشف عن.

وهنا يصف لنا طريقة سهلة وقوية للكشف عن الفسفرة Rab10 من LRRK2 على المستويات المحلية استناداً إلى منهجية العلامة ف. لأن جسم المتوفرة حاليا ضد Rab10 فوسفوريلاتيد يعمل فقط مع البروتينات overexpressed15، هذا الأسلوب استخدام العلامة ف الحزب الديمقراطي الصربي صفحة هو السبيل الوحيد لتقييم مستويا...

PD واحدة من الأمراض الأكثر شيوعاً التي الأعصاب، يغلب التي تؤثر على الخلايا العصبية تتميز في midbrain، أسفر عن الخلل في أنظمة المحركات في كبار السن1. بينما يطور معظم المرضى PD بطريقة متقطعة، وهناك أسر وراثة المرض. الطفرات في الجينات عدة وجدت لتكون مرتبطة مع FPD2. من الجينات المسببة ل FPD، LRRK2، التي كانت ثمانية مغلطه الطفرات (N1437H و R1441C/G/H/S، Y1699C، G2019S و I2020T) ترتبط FPD المهيمن موروثة التي تسمى PARK8 حتى الآن عن3،،من45. أيضا حددت العديد من الدراسات على نطاق الجينوم جمعية (جواس) متفرقة PD المرضى الاختلافات الجينية في محور LRRK2 كعامل خطر لشعبة المشتريات، مما يوحي بأن الشذوذ في وظيفة LRRK2 سبب شائع نيوروديجينيريشن في كل متفرقة و PARK8 FPD6،،من78.
LRRK2 بروتين كبير (الأحماض الأمينية 2,527) تتكون من مجال تكرار الغنية leucine، Ras غتب-ملزم للبروتينات المعقدة (ROC) المجال، ج-محطة ROC (COR) المجال والمجال كيناز البروتين سيرين/ثريونين، و مجال تكرار WD409. قم بتحديد موقع الطفرات FPD الثمانية في هذه المجالات الوظيفية؛ N1437H و R1441C/G/H/S في المجال ROC، Y1699C في المجال COR، G2019S و I2020T في مجال كيناز. منذ الطفرة G2019S، الذي يوجد أغلب الأحيان طفرة في PD المرضى10،،من1112، يزيد من نشاط كيناز LRRK2 من 2-3 إضعاف في المختبر13، هو الافتراض بأنه أن الزيادة غير طبيعية في الفسفرة LRRK2 substrate(s) سامة للخلايا العصبية. ومع ذلك، كان من المستحيل لدراسة ما إذا كان يتم تبديل الفسفرة من الناحية الفسيولوجية ذات الصلة LRRK2 ركائز في الأسرية/متفرقة PD المرضى بسبب عدم وجود أساليب تقييم ذلك في عينات المرضى المشتقة.
عموما الكشف عن الفسفرة البروتين بواسطة immunoblotting أو المرتبط بالانزيم المرتبط بالانزيم (إليزا) استخدام الأجسام المضادة على وجه الخصوص الاعتراف بدولة فوسفوريلاتيد للبروتينات أو بواسطة التحليل الشامل والمطيافيه. ومع ذلك، الاستراتيجية السابقة في بعض الأحيان لا يمكن تطبيق بسبب الصعوبات في إيجاد الأجسام المضادة الفسفرة محددة. وسم الأيضية للخلايا مع الفوسفات المشعة خيار آخر فحص مستويات الفيزيولوجية من الفسفرة عندما لا تكون الأجسام المضادة الفسفرة محددة متاحة بسهولة. بيد أنه يتطلب كمية كبيرة من المواد المشعة، وذلك ينطوي على بعض المعدات المتخصصة للإشعاع14. التحليل الشامل والمطيافيه هي أكثر حساسية بالمقارنة مع هذه الأساليب عن وأصبحت شعبية في تحليل البروتين الفسفرة. بيد أن إعداد عينة مضيعة للوقت ومكلفة الصكوك مطلوبة من أجل التحليل.
مجموعة فرعية من جتباسي رب الأسرة بما في ذلك Rab10 و Rab8 أفيد مؤخرا كركائز الفسيولوجية المباشرة ل LRRK2 استناداً إلى نتيجة ل تحليل واسع النطاق فوسفوبروتيوميك15. ثم أثبتنا أن الفسفرة Rab10 زيد ب FPD الطفرات في الخلايا الليفية الجنينية الماوس وفي رئات الفئران16knockin. وفي هذا التقرير، اخترنا أن توظف صوديوم دوديسيل كبريتات polyacrylamide هلام استشراد (الحزب الديمقراطي الصربي صفحة)-على أساس الأسلوب الذي جزيء فعلامه مبلمرة اشتركت في الحزب الديمقراطي الصربي صفحة المواد الهلامية (فالوسم الحزب الديمقراطي الصربي صفحة) للكشف عن مستويات الذاتية الفسفرة Rab10، لأنه كان لا يزال يفتقر إلى جسم حساسة للغاية محددة ل Rab10 فوسفوريلاتيد. ونحن قد أخفقت في الكشف عن الفسفرة Rab8 الذاتية بسبب الانتقائية الفقراء من الأجسام المضادة المتاحة حاليا لمجموع Rab8. ولذلك، قررنا أن نركز على الفسفرة Rab10. LRRK2 فوسفوريلاتيس Rab10 في Thr73 مكان في منتصف المنطقة المصانة عاليا "التبديل الثاني". الحفظ عالية من مواقع الفسفرة بين البروتينات Rab قد يكون واحداً من الأسباب التي تجعل الأجسام المضادة فوسفوسبيسيفيك الاعتراف بالبروتينات Rab متميزة صعبة لجعل.
الفسفرة Rab8A حسب LRRK2 يحول دون ربط Rabin8، عامل تبادل النوكليوتيدات جوانين (مرفق البيئة العالمية) الذي ينشط Rab8A عن طريق تبادل منضم الناتج المحلي الإجمالي مع أنشئ15. كما يمنع الفسفرة Rab10 و Rab8A ب LRRK2 ربط مثبطات الانفصال من الناتج المحلي الإجمالي (جديس)، التي تعتبر أساسية لتفعيل Rab البروتينات عن طريق استخراج البروتينات Rab الناتج المحلي الإجمالي زمنياً من الأغشية15. مجتمعة، هو الافتراض بأن الفسفرة Rab البروتينات التي LRRK2 يمنعهم من التنشيط على الرغم من أن الآلية الجزيئية الدقيقة والآثار الفسيولوجية الفسفرة ما زالت غير واضحة.
فالوسم الحزب الديمقراطي الصربي صفحة اخترعها كينوشيتا et al. في عام 2006: في هذا الأسلوب، اقترن الاكريلاميد تساهمي مع فالعلامه، والهلام جزيء التقاط الفوسفات مع تقارب عالية، الذي كوبوليميريزيد في صحيفة بيانات السلامة-الصفحة17. لأن الجزيئات فالعلامه في جل الحزب الديمقراطي الصربي صفحة تؤخر بشكل انتقائي التنقل الغرواني الكهربي للبروتينات فوسفوريلاتيد، فالوسم الحزب الديمقراطي الصربي صفحة يمكن فصل البروتينات فوسفوريلاتيد من غير فوسفوريلاتيد منها (الشكل 1). إذا كان هو فوسفوريلاتيد البروتين من الفائدة على بقايا متعددة، سيجري الاحتفال بسلم للفرق المقابلة لأشكال خلطات فوسفوريلاتيد. وفي حالة Rab10، نلاحظ الفرقة تحول واحد فقط، مما يشير إلى أن Rab10 هو فوسفوريلاتيد فقط في Thr73. الميزة الرئيسية للعلامة ف الحزب الديمقراطي الصربي صفحة على مدى إيمونوبلوتينج مع الأجسام المضادة الفسفرة محددة أن Rab10 فوسفوريلاتيد ويمكن الكشف عنها بواسطة إيمونوبلوتينج مع الأجسام المضادة غير الفسفرة محددة (أيالاعتراف مجموع Rab10) بعد نقله في الأغشية، وهو عموما أكثر تحديداً، وحساسة، والمتاحة من المصادر التجارية/الأكاديمية. وهناك ميزة أخرى لاستخدام العلامة ف الحزب الديمقراطي الصربي صفحة أنه يمكنك الحصول على تقدير تقريبي ستويتشيوميتري الفسفرة، التي من المستحيل إيمونوبلوتينج مع الأجسام المضادة الفسفرة محددة أو بوصفها الأيضية للخلايا مع المشعة الفوسفات.
وبصرف النظر عن الاستخدام غير مكلفة فالعلامه الاكريلاميد وبعض التعديلات الطفيفة المتعلقة به، يتبع الأسلوب الحالي للكشف عن الفسفرة Rab10 من LRRK2 عامة بروتوكول إيمونوبلوتينج.ولذلك، ينبغي واضحة وقابلة للتنفيذ بسهولة في أي المختبرات حيث إيمونوبلوتينج ممارسة معتادة، مع أي من أنواع العينات بما في ذلك البروتينات المنقاة، ليساتيس الخلية والأنسجة هوموجيناتيس.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>homemade</td>
			<td></td>
			<td>150 mM NaCl, 8 mM Na2HPO4-12H2O, 2.7 mM KCl, 1.5 mM KH2PO4 in MilliQ water and sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Nacalai Tesque</td>
			<td>31320-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Disodium Hydrogenphosphate 12-Water</td>
			<td>Wako</td>
			<td>196-02835</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Wako</td>
			<td>163-03545</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate</td>
			<td>Wako</td>
			<td>169-04245</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsin (10X)</td>
			<td>Sigma-Aldrich</td>
			<td>T4549</td>
			<td>Dilute 10-fold with sterile DPBS for preparing working solution</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium 
			(DMEM)</td>
			<td>Wako</td>
			<td>044-29765</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>BioWest</td>
			<td>S1560</td>
			<td>Heat-inactivated at 56 &#176;C for 30 min</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>Wako</td>
			<td>168-23191</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Wako</td>
			<td>342-01375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Wako</td>
			<td>198-13765</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine HCl MAX, Linear, Mw 40,000 (PEI MAX 40000)</td>
			<td>PolySciences, Inc.</td>
			<td>24765-1</td>
			<td>Stock solution was prepared in 20 mM HEPES-NaOH pH 7.0 at 1 mg/mL and the pH was then adjusted to 7.0 with NaOH</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Wako</td>
			<td>045-28335</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>STAR</td>
			<td>RSP-THA500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Wako</td>
			<td>080-01066</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene(10) Octylphenyl Ether</td>
			<td>Wako</td>
			<td>160-24751</td>
			<td>Equivalent to Triton X-100</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8217;,N&#8217;-tetraacetic acid (EGTA)</td>
			<td>Wako</td>
			<td>346-01312</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium orthovanadate(V)</td>
			<td>Wako</td>
			<td>198-09752</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluoride</td>
			<td>Kanto Chemical</td>
			<td>37174-20</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Glycerophosphoric Acid Disodium Salt Pentahydrate</td>
			<td>Nacalai Tesque</td>
			<td>17103-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate decahydrate</td>
			<td>Kokusan Chemical</td>
			<td>2113899</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcystin-LR</td>
			<td>Wako</td>
			<td>136-12241</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako</td>
			<td>196-00015</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA-free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Dissolve one tablet in 1 mL water, which can be stored at -20 &#176;C for a month. Use it at 1:50 dilution for cell lysis</td>
		</tr>
		<tr>
			<td>Pierce Coomassie (Bradford) Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Nacalai Tesque</td>
			<td>31607-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Wako</td>
			<td>075-00616</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Wako</td>
			<td>021-02911</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Kanto Chemical</td>
			<td>25099-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Wako</td>
			<td>056-06967</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Wako</td>
			<td>136-01837</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-binding tag acrylamide</td>
			<td>Wako</td>
			<td>AAL-107</td>
			<td>P-tag acrylamide</td>
		</tr>
		<tr>
			<td>40% (w/v) acrylamide solution</td>
			<td>Nacalai Tesque</td>
			<td>06119-45</td>
			<td>Acrylamide:Bis = 29:1</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Nacalai Tesque</td>
			<td>33401-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Wako</td>
			<td>016-08021</td>
			<td>10% (w/v) solution was prepared by dissolving the powder of ammonium persulfate in MilliQ water</td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Wako</td>
			<td>166-04831</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese chloride tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M3634</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Prestained Standard</td>
			<td>Bio-Rad</td>
			<td>1610374, 1610373, 1610377</td>
			<td>Molecular weight marker used in the protocol</td>
		</tr>
		<tr>
			<td>WIDE-VIEW Prestained Protein Size Marker III</td>
			<td>Wako</td>
			<td>230-02461</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Nacalai Tesque</td>
			<td>17109-64</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Protran NC 0.45</td>
			<td>GE Healthcare</td>
			<td>10600007</td>
			<td>Nitrocellulose membrane</td>
		</tr>
		<tr>
			<td>Durapore Membrane Filter</td>
			<td>EMD Millipore</td>
			<td>GVHP00010</td>
			<td>PVDF membrane</td>
		</tr>
		<tr>
			<td>Filter Papers No.1</td>
			<td>Advantec</td>
			<td>00013600</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>Nacalai Tesque</td>
			<td>28322-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Wako</td>
			<td>017-00251</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>polyoxyethylenesorbitan monolaurate</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Wako</td>
			<td>345-01865</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Difco Laboratories</td>
			<td>232100</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunostar</td>
			<td>Wako</td>
			<td>291-55203</td>
			<td>ECL solution (Normal sensitivity)</td>
		</tr>
		<tr>
			<td>Immunostar LD</td>
			<td>Wako</td>
			<td>290-69904</td>
			<td>ECL solution (High sensitivity)</td>
		</tr>
		<tr>
			<td>CBB staining solution</td>
			<td>homemade</td>
			<td></td>
			<td>1 g CBB R-250, 50% (v/v) methanol, 10% (v/v) acetic acid in 1 L of MilliQ water</td>
		</tr>
		<tr>
			<td>CBB R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td></td>
		</tr>
		<tr>
			<td>CBB destaining solution</td>
			<td>homemade</td>
			<td></td>
			<td>12% (v/v) methanol, 7% (v/v) acetic acid in 1 L MilliQ water</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-HA antibody</td>
			<td>Sigma-Aldrich</td>
			<td>11583816001</td>
			<td>Used at 0.2 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>anti-Rab10 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>#8127</td>
			<td>Used at 1:1000 for immunoblotting. 
			Specificity was confirmed by CRISPR KO in Ito et al., Biochem J, 2016.</td>
		</tr>
		<tr>
			<td>anti-pSer935 antibody</td>
			<td>Abcam</td>
			<td>ab133450</td>
			<td>Used at 1 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>anti-LRRK2 antibody</td>
			<td>Abcam</td>
			<td>ab133518</td>
			<td>Used at 1 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>anti-&#945;-tubulin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T9026</td>
			<td>Used at 1 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>anti-GAPDH antibody</td>
			<td>Santa-Cruz</td>
			<td>sc-32233</td>
			<td>Used at 0.02 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Sheep Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>515-035-003</td>
			<td>Used at 0.16 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-035-003</td>
			<td>Used at 0.16 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Inhibitors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSK2578215A</td>
			<td>MedChem Express</td>
			<td>HY-13237</td>
			<td>Stock solution was prepared in DMSO at 10 mM and stored at -80 &#176;C</td>
		</tr>
		<tr>
			<td>MLi-2</td>
			<td>Provided by Dr Dario Alessi (University of Dundee)</td>
			<td></td>
			<td>Stock solution was prepared in DMSO at 10 mM and stored at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rab10/pcDNA5 FRT TO HA</td>
			<td>Provided by Dr Dario Alessi 
			(University of Dundee)</td>
			<td></td>
			<td>This plasmid expresses amino-terminally HA-tagged human Rab10.</td>
		</tr>
		<tr>
			<td>LRRK2 WT/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Ito et al., Biochemistry, 46: 1380&#8211;1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged wild-type human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 K1906M/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Ito et al., Biochemistry, 46: 1380&#8211;1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged K1906M kinase-inactive mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 N1437H/p3xFLAG-CMV-10</td>
			<td></td>
			<td></td>
			<td>This paper. This plasmid expresses amino-terminally 3xFLAG-tagged N1437H FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 R1441C/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441C FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 R1441G/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441G FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 R1441H/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441H FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 R1441S/p3xFLAG-CMV-10</td>
			<td></td>
			<td></td>
			<td>This paper. This plasmid expresses amino-terminally 3xFLAG-tagged R1441S FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 Y1699C/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged Y1699C FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 G2019S/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged G2019S FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 I2020T/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged I2020T FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Forma Series II 3110 Water-Jacketed</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto Pipette</td>
			<td>Drummond</td>
			<td>Pipet-Aid PA-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette P10</td>
			<td>Nichiryo</td>
			<td>00-NPX2-10</td>
			<td>0.5&#8211;10 &#956;L</td>
		</tr>
		<tr>
			<td>Micropipette P200</td>
			<td>Nichiryo</td>
			<td>00-NPX2-200</td>
			<td>20&#8211;200 &#956;L</td>
		</tr>
		<tr>
			<td>Micropipette P1000</td>
			<td>Nichiryo</td>
			<td>00-NPX2-1000</td>
			<td>100&#8211;1000 &#956;L</td>
		</tr>
		<tr>
			<td>Tips for micropipette P10</td>
			<td>STAR</td>
			<td>RST-481LCRST</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Tips for micropipette P200</td>
			<td>FUKAEKASEI</td>
			<td>1201-705YS</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Tips for micropipette P1000</td>
			<td>STAR</td>
			<td>RST-4810BRST</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>5 mL disporsable pipette</td>
			<td>Greiner</td>
			<td>606180</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>10 mL disporsable pipette</td>
			<td>Greiner</td>
			<td>607180</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>25 mL disporsable pipette</td>
			<td>Falcon</td>
			<td>357535</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Hematocytometer</td>
			<td>Sunlead Glass</td>
			<td>A126</td>
			<td>Improved Neubeuer</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>AGC Techno Glass</td>
			<td>3810-006</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries</td>
			<td>Vortex-Genie 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Sumitomo Bakelite</td>
			<td>MS-93100</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>STAR</td>
			<td>RSV-MTT1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>AGC Techno Glass</td>
			<td>2323-015</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>AGC Techno Glass</td>
			<td>2343-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuges</td>
			<td>TOMY</td>
			<td>MX-307</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Greiner</td>
			<td>655061</td>
			<td>Not for tissue culture</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M2e</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS&#8211;PAGE tanks</td>
			<td>Nihon Eido</td>
			<td>NA-1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer tanks</td>
			<td>Nihon Eido</td>
			<td>NA-1510B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel plates (notched)</td>
			<td>Nihon Eido</td>
			<td>NA-1000-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel plates (plain)</td>
			<td>Nihon Eido</td>
			<td>NA-1000-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon spacers</td>
			<td>Nihon Eido</td>
			<td>NA-1000-16</td>
			<td></td>
		</tr>
		<tr>
			<td>17-well combs</td>
			<td>Nihon Eido</td>
			<td>Custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>Binder clips</td>
			<td>Nihon Eido</td>
			<td>NA-1000-15</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Terumo</td>
			<td>SS-05SZ</td>
			<td></td>
		</tr>
		<tr>
			<td>21G</td>
			<td>Terumo</td>
			<td>NN-2138R</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Station 1000 VC</td>
			<td>ATTO</td>
			<td>AE-8450</td>
			<td>Power supply for SDS&#8211;PAGE and transfer</td>
		</tr>
		<tr>
			<td>Large weighing boats</td>
			<td>Ina Optika</td>
			<td>AS-DL</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic containers</td>
			<td>AS ONE</td>
			<td>PS CASE No.4</td>
			<td>10 x 80 x 50 mm</td>
		</tr>
		<tr>
			<td>Rocking shaker</td>
			<td>Titech</td>
			<td>NR-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrene foam box</td>
			<td>generic</td>
			<td></td>
			<td>The internal dimensions should fit one transfer tank (200 x 250 x 250 mm).</td>
		</tr>
		<tr>
			<td>ImageQuant LAS-4000</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>An imager equipped with a cooled CCD camera for detection of ECL</td>
		</tr>
	</tbody>
</table>

rab10 phosphorylation detection by lrrk2 activity using sds-page with a phosphate-binding tag

الطفرات في الغنية لوسين تكرار كيناز 2 (LRRK2) تبين أن تكون مرتبطة بمرض باركنسون الأسرية (FPD). منذ التنشيط غير طبيعي لنشاط كيناز LRRK2 قد تورط في الآلية المرضية لشعبة المشتريات، من الضروري وضع طريقة تقييم مستويات الفسيولوجية للنشاط كيناز من LRRK2. وكشفت الدراسات التي أجريت مؤخرا أن LRRK2 فوسفوريلاتيس أفراد الأسرة Rab جتباسي، بما في ذلك Rab10، في ظل الظروف الفسيولوجية. على الرغم من الفسفرة Rab10 الذاتية التي LRRK2 في الخلايا المستزرعة يمكن الكشف عنها بواسطة الكتلي، كان من الصعب أن الكشف عن ذلك إيمونوبلوتينج وبسبب حساسية أجسام محددة الفسفرة المتاحة حاليا للفقراء Rab10. وهنا، يمكننا وصف بسيط الأسلوب للكشف عن مستويات الفسفرة Rab10 من LRRK2 الذاتية استناداً إلى إيمونوبلوتينج استخدام الصوديوم دوديسيل كبريتات polyacrylamide هلام التفريد (الحزب الديمقراطي الصربي صفحة) جنبا إلى جنب مع علامة ربط الفوسفات (P--علامة)، التي ن-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N،ن '،ن'-تريس (بيريدين-2-يل-الميثيل) ديامينوبروبان-1، 3--2-الرتب الأخرى. هذا البروتوكول لا يشكل مثالاً للمنهجية التي تستخدم علامة ف فحسب بل يمكن أيضا تقييم كيف يغير الطفرات، فضلا عن العلاج المانع والإدارة أو أي عوامل أخرى مما يشير إلى المصب من LRRK2 في الخلايا والأنسجة .

نظام أوفيريكسبريشن: الفسفرة من ها Rab10 3 × العلم-LRRK2 في HEK293 الخلايا:
تم transfected الخلايا HEK293 مع 0.266 ميكروغرام من Rab10 هكتار البرية من نوع و 1.066 ميكروغرام من 3 × العلم-LRRK2 (المسخ كيناز نشط، والبرية من نوع (K1906M)، أو طفرات FPD). بحثت الفسفرة Rab10 فالوسم الحزب ...

Watch this Scientific Journal Video about Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag at JoVE.com

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

هذه الدراسة توضح طريقة بسيطة للكشف عن مستويات الذاتية من الفسفرة Rab10 بالغنية لوسين تكرار كيناز 2.

كشف الفسفرة Rab10 بالنشاط LRRK2 باستخدام مخزونات النشر الاستراتيجي-الصفحة مع علامة ربط الفوسفات

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

rab10-phosphorylation-detection-lrrk2-activity-using-sds-page-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

14.8K Views.  The University of Tokyo. هذه الدراسة توضح طريقة بسيطة للكشف عن مستويات الذاتية من الفسفرة Rab10 بالغنية لوسين تكرار كيناز 2.

Video: Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

حساسة للغاية وسريع كشف الإسفار مع محلل الحنق المحمولة

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

الكشف عن تعتمد على درجة الحموضة آخر من<em&gt; كولاي</em&gt; الوصيفة HdeB<em&gt; في المختبر</em&gt; و<em&gt; في فيفو</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

فحص البروتين كيناس النشاط مع راديولابيلد أتب

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

أوليغوببتيد مسابقة الفحص لتحديد موقع الفسفرة

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

بوليميراز الدنا نشاط الإنزيم استخدام القريبة من الأشعة تحت الحمراء الحمض النووي المسمى الفلورسنت تصور بالتفريد هلام الاكريلاميد

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

الكشف عن برينيليشن جتباسي صغيرة وأنشئ ملزمة باستخدام تجزئة غشاء ومقايسة الممتز GTPase مرتبطة

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

تحديد كيناز Cyclin تعتمد على 1 مواقع الفسفرة محددة من قبل كيناز في المختبر الاعتداء

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

الكشف عن نشاط ج فوسفوليباسي في هوموجيناتي المخ من نحل العسل

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

انجذاب تطهير المجمعات البروتين ترانسلوكون بلاستيدات الخضراء باستخدام علامة الحنفية

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

كشف الفسفرة Rab10 بالنشاط LRRK2 باستخدام مخزونات النشر الاستراتيجي-الصفحة مع علامة ربط الفوسفات

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