עבודה זו נתמכה על ידי NIH מענקים RO1 DC006497, RO1 NS057289 ו PO1 ES016738 (עד ז&#39;ז&#39;אנג); קליפורניה המכון לרפואה הרגנרציה מענקים RS1-00331-1 ו-RL1 00682-1 (עד ז&#39;ז&#39;אנג); הפרקינסון האמריקאי מחלות האגודה (&#39;עד ת&#39;ג&#39;אנג צ&#39;ו י&quot;ש); ואת ג&#39;יי מייקל פוקס הקרן לחקר הפרקינסון (צ&#39;אנג).

גילוי ubiquitination חלבון בתאים בתרבית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תאים בתרבית Transfect עם פלסמידים המבטאים את החלבון של עניין (epitope-tagged גרסה) יוביקוויטין. </li><li style=";text-align:right;direction:rtl"> הפוך את התא להשלים תמוגה חיץ (2% SDS, 150 mM NaCl, 10 mM טריס-HCl, pH 8.0) עם orthovanadate 2mm נתרן, נתרן פלואוריד 50 מ&quot;מ, מעכבי פרוטאז. </li><li style=";text-align:right;direction:rtl"> Lyse תאים עם 100 חיץ תמוגה μl תא לכל צלחת (6 צלחת ס&quot;מ). אם מנה גדולה יותר משמש, לכוון את עצמת השמע בהתאם. מערבולת התבשיל בזהירות לתת החיץ תמוגה לכסות את כל השטח של תאים בוגרים. </li><li style=";text-align:right;direction:rtl"> איסוף התאים עם מגרד תא ולהעביר את lysates תא לתוך צינור Eppendorf 1.5 מ&quot;ל. מניחים את הצינור לצלחת חמה מיד לרתיחה 10 דקות. </li><li style=";text-align:right;direction:rtl"> שאר התאים עם מכשיר sonication. </li><li style=";text-align:right;direction:rtl"> הוסף 900ul של חיץ דילול (10 mM טריס-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, טריטון 1%). דגירה דגימות 4 מעלות צלזיוס במשך 30-60 דקות עם רוטציה. </li><li style=";text-align:right;direction:rtl"> ספין דגימות מדולל ב XG 20,000 למשך 30 דקות. מעבירים את supernatant שנוצר צינור Eppendorf חדש. היזהר לא להפריע גלולה. </li><li style=";text-align:right;direction:rtl"> למדוד את ריכוז החלבון. </li><li style=";text-align:right;direction:rtl"> הכינו חלבון או G-agarose חרוז מצומדות נוגדנים נגד חלבון המטרה במאגר תואם (50% slurry). חותכים את הקצה הצר של טיפ P-200 פיפטה ולהעביר 14-20 μl של שרף מיקרוגרם של 500-1,500 lysates תא מוכנים immunoprecipitation. עבור תאים עם נצילות גבוהה transfection, 500 מיקרוגרם יהיה מספיק. עבור תאים עם נצילות transfection נמוך, יותר חלבון עשוי להיות נחוץ. </li><li style=";text-align:right;direction:rtl"> דגירה את התערובת lysate-חרוז התא ב 4 ° C לילה עם רוטציה. </li><li style=";text-align:right;direction:rtl"> ספין למטה חרוזים XG ב 5000 עבור 5 דקות. לשאוב supernatant. שטפו את שרף עם למאגר כביסה (10 mM טריס-HCl, pH 8.0, 1 M NaCl, 1 mM EDTA, 1% NP-40) פעמיים. </li><li style=";text-align:right;direction:rtl"> ספין החרוזים בפעם האחרונה ב XG 20,000 למשך 30 שניות. לשאוב החיץ כביסה שיורית ומרתיחים שרף עם חיץ טעינת SDS 2X. </li><li style=";text-align:right;direction:rtl"> טען דוגמאות על ג&#39;ל SDS עמודים עבור immunoblotting ניתוח. </li><li style=";text-align:right;direction:rtl"> זיהוי היוביקוויטין לבין חלבון המטרה עם נוגדנים בהתאמה. עבור immunoblotting, אנחנו בדרך כלל לזהות היוביקוויטין הראשון. הממברנה מכן ניתן יהיה להשתמש כדי לזהות את זירז חלבון. </li></ol> זיהוי של חלבון המטרה ubiquitination על דרך ב assay ubiquitination חוץ גופית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפוך את החיץ ubiquitination 5X (100 מ&quot;מ טריס-HCl, pH 7.5, 25 מ&quot;מ MgCl2, 2.5 מ&quot;מ DTT, 10 mM ATP). אחסן אותם aliquots קטן -20 ° C עד חודש 6. פרוטוקולים מסוימים גם להוסיף פוספט קריאטין קריאטין קינז לתוך למאגר עבור 1,2 התחדשות ATP. </li><li style=";text-align:right;direction:rtl"> עבור כל תגובה, להכין תערובת המכילה את הפעולות הבאות: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> Μl 8 </td><td> Ubiquitination 5X חיץ </td></tr><tr><td> 250 ng </td><td> ubiquitination E1 </td></tr><tr><td> 500 ng </td><td> ubiquitination E2 </td></tr><tr><td> 0.5 מיקרוגרם </td><td> היוביקוויטין </td></tr><tr><td> 0.5 מיקרוגרם </td><td> חלבון עניין </td></tr><tr><td></td><td> נפח המים הכולל 40 μl </td></tr></tbody></table> כמו שולטת, להכין תגובות דומות בהעדר או E1, E2, או יוביקוויטין. </li><li style=";text-align:right;direction:rtl"> דגירה את התערובת על 37 מעלות צלזיוס למשך 1 שעה או יותר. </li><li style=";text-align:right;direction:rtl"> עצור את התגובה על ידי הוספת SDS-PAGE חיץ מדגם להרתיח את המדגם עבור 10 דקות. </li><li style=";text-align:right;direction:rtl"> טען דוגמאות על ג&#39;ל SDS-PAGE עבור immunoblotting ניתוח. </li><li style=";text-align:right;direction:rtl"> זיהוי היוביקוויטין לבין חלבון המטרה עם נוגדנים בהתאמה. </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.

במצגת זו, אנו הראשון שתיאר צעדים המאפשרים לזהות שינוי יוביקוויטין על החלבון של עניין בתאי יונקים בתרבית. על מנת לזהות ubiquitination במיוחד על החלבון של עניין, לא על הלא קוולנטית חלבונים אינטראקציה, היינו מצב מחמירים עבור תמוגה התא, immunoprecipitation ושטיפת 1,3. Ubiquitination, זוהה ע...

detection of protein ubiquitination

Ubiquitination, את הקובץ המצורף קוולנטיים של היוביקוויטין פוליפפטיד למקד חלבונים, הוא שינוי posttranslational המפתח בוצע על ידי קבוצה של שלושה אנזימים. הם כוללים היוביקוויטין, הפעלת אנזים E1, E2 היוביקוויטין-conjugating האנזים, האנזים E3 יוביקוויטין. בניגוד ל E1 ו E2, E3 המצע ligases היוביקוויטין סגוליות לתצוגה. מצד שני, אנזימים deubiquitylating יש תפקידים רבים בעיבוד חלבונים polyubiquitinated. Ubiquitination יכול לגרום לשינוי של יציבות חלבון, לוקליזציה הסלולר, ופעילות ביולוגית. מוטציות של גנים המעורבים במסלול ubiquitination / deubiquitination או תפקוד מערכת היוביקוויטין שינו המשויכים רבים מחלות אנושיות שונות, כגון סוגים שונים של סרטן, ניוון מוחיים, והפרעות מטבוליות. הגילוי של ubiquitination שינה או נורמלי של חלבונים היעד עשויה לספק הבנה טובה יותר על בפתוגנזה של מחלות אלה. כאן אנו מתארים פרוטוקולים לזהות ubiquitination החלבון בתאים בתרבית<em&gt; In vivo</em&gt; ו מבחנות<em&gt; במבחנה</em&gt;. פרוטוקולים אלה שימושיים גם כדי לזהות שינויים אחרים היוביקוויטין כמו מולקולה קטנה כגון sumolyation ו neddylation.

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

Detection of Protein Ubiquitination

Ubiquitination הוא שינוי posttranslational המפתח בוצע על ידי קבוצה של שלושה אנזימים. מוטציות של גנים המעורבים שינוי זה קשור הרבה מחלות אנושיות שונות. כאן אנו מתארים פרוטוקולים לזהות ubiquitination החלבון בתאים בתרבית<em&gt; In vivo</em&gt; ו מבחנות<em&gt; במבחנה</em&gt;.

זיהוי של חלבון Ubiquitination

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

Research

JoVE Journal

Biology

42.6K Views.  The Sanford Burnham Institute for Medical Research. Ubiquitination הוא שינוי posttranslational המפתח בוצע על ידי קבוצה של שלושה אנזימים. מוטציות של גנים המעורבים שינוי זה קשור הרבה מחלות אנושיות שונות. כאן אנו מתארים פרוטוקולים לזהות ubiquitination החלבון בתאים בתרבית In vivo ו מבחנות במבחנה.

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.

אצווה Immunostaining לגילוי בקנה מידה גדול חלבון הנמצא במוח הקופים שלמים

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.

הכתמה של חלבונים עם ג&#39;ל Coomassie G-250 ללא חומצה אצטית ממס אורגני

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.

באמצעות מערכת GELFREE 8100 ההיפוך חלוקה על ההיפוך חלוקה משקל מבוסס מולקולרית עם שלב החילוץ נוזלי

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.

In-vivo גילוי של חלבונים אינטראקציות על מיקרו-בדוגמת משטחים

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.

איתור של אינטראקציות חלבון הצמח באמצעות bimolecular תואם Gateway הקרינה השלמה (BiFC) מערכת

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.

הקרנת siRNA לזיהוי יוביקוויטין ורגולטורים מערכת דמויות יוביקוויטין של מסלולים ביולוגיים בתאי יונקים בתרבית

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.

הקרנה ירוקה פלורסנט מבוסס חלבון ביטוי של החלבונים ממברנה ב<em&gt; Coli Escherichia</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.

טכניקת טיהור חלבון המאפשרת איתור של Sumoylation וUbiquitination של שמרים להנץ החלבונים קינטוכור Ndc10 וNdc80

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.

איתור של הפנמת קולטן מצמיד חלבון G-מופעל ליגנד ידי מיקרוסקופי Confocal

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.

אין ויוו זיהוי וניתוח של SUMOylation חלבון Rb בתאי אדם

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