وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح RO1 DC006497 ، RO1 NS057289 وPO1 ES016738 (الألف إلى الياء تشانغ) ؛ معهد كاليفورنيا للحصول على منح الطب التجديدي RS1 - 00331-1 وRL1 - 00682 - 1 (الألف إلى الياء تشانغ) ، وباركنسون الأمريكية رابطة مرض (الألف إلى الياء تشو تشانغ وYS) ، وجيه مايكل فوكس مؤسسة للأبحاث مرض باركنسون (Z. تشانغ).

اكتشاف بروتين في الخلايا ubiquitination مثقف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مثقف خلايا Transfect البلازميدات مع التعبير عن المصالح والبروتين (حاتمة الموسومة إصدار) اليوبيكويتين. </li><li style=";text-align:right;direction:rtl"> جعل كاملة العازلة تحلل الخلية (2 ٪ SDS ، 150 مم كلوريد الصوديوم ، و 10 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 8.0) مع orthovanadate 2mm والصوديوم ، و 50 ملي فلوريد الصوديوم ، ومثبطات الأنزيم البروتيني. </li><li style=";text-align:right;direction:rtl"> ليز الخلايا مع 100 ميكرولتر تحلل الخلية العازلة في لوحة (6 طبق سم). إذا تم استخدام أكبر صحن ، وضبط حجم وفقا لذلك. دوامة الطبق بعناية للسماح للتحلل العازلة تغطية المنطقة بأكملها من الخلايا المزروعة. </li><li style=";text-align:right;direction:rtl"> جمع الخلايا مع مكشطة الخلية ونقل lysates الخلية في أنبوب 1.5 مل إيبندورف. وضع أنبوب على صفيحة ساخنة على الفور ليغلي لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> قص الخلايا مع جهاز صوتنة. </li><li style=";text-align:right;direction:rtl"> إضافة 900ul العازلة للتخفيف (10 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 8.0 ، 150 مم كلوريد الصوديوم ، EDTA 2 مم ، تريتون 1 ٪). احتضان العينات في درجة مئوية (4) ل30-60 دقيقة مع التناوب. </li><li style=";text-align:right;direction:rtl"> تدور العينات المخففة في XG 20000 لمدة 30 دقيقة. طاف نقل الناتج إلى أنبوب إيبندورف جديدة. يجب الحرص على عدم تعكير صفو بيليه. </li><li style=";text-align:right;direction:rtl"> قياس تركيز البروتين. </li><li style=";text-align:right;direction:rtl"> يعد البروتين A - G - agarose أو حبة مترافق الأضداد ضد البروتين المستهدف في منطقة عازلة متوافق (50 ٪ الطين). قطع نهاية ضيقة من طرف P - 200 ماصة ونقل 14-20 ميكرولتر من الراتنج ل500-1،500 ميكروغرام من lysates الخلية التي أعدت للمناعي. لخلايا ذات كفاءة عالية ترنسفكأيشن ، سوف يكون كافيا 500 ميكروغرام. للخلايا مع انخفاض كفاءة ترنسفكأيشن ، قد تكون هناك حاجة إلى مزيد من البروتين. </li><li style=";text-align:right;direction:rtl"> احتضان الخلية lysate - حبة الخليط في 4 درجات مئوية خلال الليل مع التناوب. </li><li style=";text-align:right;direction:rtl"> تدور باستمرار الخرز في 5000 x ج لمدة 5 دقائق. نضح لطاف. غسل راتنج مع الغسيل العازلة (10 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 8.0 ، 1 م كلوريد الصوديوم ، EDTA 1 ملم ، 1 ٪ NP - 40) مرتين. </li><li style=";text-align:right;direction:rtl"> تدور حبات لمرة أخيرة في XG 20000 لمدة 30 ثانية. نضح المخزن الغسيل المتبقية وتغلي الراتنج مع العازلة 2X تحميل SDS. </li><li style=";text-align:right;direction:rtl"> تحميل عينات على جل SDS - PAGE immunoblotting لتحليلها. </li><li style=";text-align:right;direction:rtl"> اكتشاف بروتين اليوبيكويتين والهدف مع الأضداد منها. لimmunoblotting ، ونحن عموما كشف اليوبيكويتين الأولى. وعندئذ يكون الغشاء المستخدمة للكشف عن البروتين عجل. </li></ol> اكتشاف بروتين ubiquitination على الهدف عن طريق فحص في المختبر ubiquitination <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل 5X ubiquitination العازلة (100 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 7.5 ، 25 مم MgCl2 ، 2.5 ملي DTT ، 10 ملي ATP). تخزينها في aliquots صغيرة في -20 درجة مئوية لمدة تصل إلى 6 أشهر. بعض البروتوكولات أيضا إضافة فوسفات الكرياتين كيناز والكرياتين في المخزن المؤقت لل1،2 تجديد ATP. </li><li style=";text-align:right;direction:rtl"> لكل رد فعل ، وإعداد مزيج يحتوي على ما يلي : <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> ميكرولتر 8 </td><td> 5X ubiquitination العازلة </td></tr><tr><td> 250 نانوغرام </td><td> ubiquitination E1 </td></tr><tr><td> 500 نانوغرام </td><td> ubiquitination E2 </td></tr><tr><td> 0.5 ميكروغرام </td><td> اليوبيكويتين </td></tr><tr><td> 0.5 ميكروغرام </td><td> بروتين من الفائدة </td></tr><tr><td></td><td> المياه لحجم الكلي 40 ميكرولتر </td></tr></tbody></table> والضوابط ، وإعداد ردود فعل مماثلة في غياب أي E1 ، E2 ، أو اليوبيكويتين. </li><li style=";text-align:right;direction:rtl"> احتضان الخليط عند 37 درجة مئوية لمدة 1 ساعة أو أكثر. </li><li style=";text-align:right;direction:rtl"> وقف رد الفعل بإضافة SDS - PAGE العازلة عينة ونموذج تغلي لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> تحميل عينات على SDS - PAGE هلام immunoblotting لتحليلها. </li><li style=";text-align:right;direction:rtl"> اكتشاف بروتين اليوبيكويتين والهدف مع الأضداد منها. </li></ol>

<ol>
	<li>Xiong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1,+and+DJ-1+form+a+ubiquitin+E3+ligase+complex+promoting+unfolded+protein+degradation.">PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation.</a> J Clin Invest. 119, 650-660 (2009).</li><li>Xirodimas, D. P., Saville, M. K., Bourdon, J. C., Hay, R. T., Lane, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdm2-mediated+NEDD8+conjugation+of+p53+inhibits+its+transcriptional+activity.">Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.</a> Cell. 118, 83-97 (2004).</li><li>Didier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF5,+a+RING+finger+protein+that+regulates+cell+motility+by+targeting+paxillin+ubiquitination+and+altered+localization.">RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization.</a> Mol Cell Biol. 23, 5331-5345 (2003).</li><li>Laney, J. D., Hochsetrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unit+14.5+Analysis+of+Protein+Ubiquitination.">Unit 14.5 Analysis of Protein Ubiquitination.</a> Current Protocols in Protein Science. , 14.5.1-14.5.11 (2002).</li></ol>

The authors have nothing to disclose.

في هذا العرض ، ونحن أول من وصف الخطوات التي تسمح الكشف عن تعديل اليوبيكويتين على بروتين من الاهتمام في خلايا الثدييات مثقف. استخدمنا من أجل الكشف عن ubiquitination تحديدا على البروتين من الفائدة ، وليس على البروتينات المتفاعلة غير تساهمي ، وهو شرط صارم لتحلل الخلية ، وغسل <su...

detection of protein ubiquitination

Ubiquitination ، المرفق التساهمية من اليوبيكويتين ببتيد لاستهداف البروتينات ، هو تعديل مفتاح posttranslational التي قامت بها مجموعة من ثلاثة الانزيمات. وهي تشمل اليوبيكويتين - E1 تفعيل أنزيم اليوبيكويتين - E2 التصريف الانزيم ، واليوبيكويتين E3 يغاز. خلافا لE1 و E2 ، E3 عرض ligases اليوبيكويتين خصوصية الركيزة. من ناحية أخرى ، فإن العديد من الانزيمات deubiquitylating أدوارا في تجهيز البروتينات polyubiquitinated. يمكن أن يؤدي إلى تغيير Ubiquitination الاستقرار البروتين ، وتوطين الخلوية ، والنشاط البيولوجي. ترتبط طفرات في الجينات المسؤولة عن المسار ubiquitination / deubiquitination أو اختلال وظيفة نظام اليوبيكويتين مع العديد من الأمراض البشرية المختلفة مثل أنواع مختلفة من السرطان ، وتنكس عصبي ، والاضطرابات الأيضية. قد كشف ubiquitination تغيير أو العادي للبروتينات هدف توفير فهم أفضل عن التسبب في هذه الأمراض. هنا ، نحن تصف بروتوكولات للكشف عن البروتين في الخلايا ubiquitination مثقف<em&gt; في الجسم الحي</em&gt; وبالأنابيب<em&gt; في المختبر</em&gt;. هذه البروتوكولات هي مفيدة أيضا للكشف عن الأخرى مثل تعديل اليوبيكويتين جزيء صغير مثل sumolyation وneddylation.

Watch this Scientific Journal Video about Detection of Protein Ubiquitination at JoVE.com

Detection of Protein Ubiquitination

Ubiquitination هو تعديل مفتاح posttranslational التي قامت بها مجموعة من ثلاثة الانزيمات. ترتبط طفرات في الجينات المسؤولة عن هذا التعديل مع العديد من الأمراض البشرية المختلفة. هنا ، نحن تصف بروتوكولات للكشف عن البروتين في الخلايا ubiquitination مثقف<em&gt; في الجسم الحي</em&gt; وبالأنابيب<em&gt; في المختبر</em&gt;.

اكتشاف بروتين Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Research

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Biology

42.6K Views.  The Sanford Burnham Institute for Medical Research. Ubiquitination هو تعديل مفتاح posttranslational التي قامت بها مجموعة من ثلاثة الانزيمات. ترتبط طفرات في الجينات المسؤولة عن هذا التعديل مع العديد من الأمراض البشرية المختلفة. هنا ، نحن تصف بروتوكولات للكشف عن البروتين في الخلايا ubiquitination مثقف في الجسم الحي وبالأنابيب

Video: Detection of Protein Ubiquitination

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Immunostaining دفعي للكشف عن البروتين واسعة النطاق في الدماغ القرد الجامعة

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

تلطيخ من البروتينات في الهلام مع Coomassie G - 250 دون عضوي حمض الخليك والمذيبات

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

باستخدام نظام التقطير GELFREE 8100 لتقطير الوزن الجزيئي وبناء مع مرحلة الانتعاش السائل

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

في الجسم الحي ، الكشف عن البروتين البروتين التفاعلات على مايكرو نقوش السطوح

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

الكشف عن تفاعلات البروتين في النبات باستخدام عبارة الإسفار ثنائي الجزيء المتوافقة التكملة (BiFC) نظام

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

سيرنا فحص لتحديد اليوبيكويتين واليوبيكويتين مثل المنظمين نظام مقررات تمهيدية البيولوجية في خلايا الثدييات مثقف

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

الأخضر الفلورسنت القائم على البروتين التعبير فحص غشاء البروتينات في<em&gt; الإشريكية القولونية</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

البروتين تنقية تقنية تسمح الكشف عن Sumoylation وUbiquitination من الناشيء الخميرة الحيز الحركي البروتينات Ndc10 وNdc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

الكشف عن تنشيط يجند G-إلى جانب البروتين مستقبلات التطبع التي كتبها متحد البؤر المجهري

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

في فيفو كشف وتحليل م ع سومويليشن البروتين في الخلايا البشرية

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...