עבודה זו נתמכה בחלקה על ידי מענק קרנות לתשתיות למחקר (RSFG) מאוניברסיטת אינדיאנה-Purdue University, אינדיאנפוליס, ובחלקה על ידי בפרס של איגוד הלב האמריקאי 14GRNT20390154, כדי ARK

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The authors have nothing to disclose.

אנו מדגימים את היתרון של גישה משולבת לניתוח הרכבה הפרוטאזום ידי nondenaturing עמוד באמצעות פרוטאזומים archaeal רקומביננטי. 9,11 השיטה הרגילה של Coexpression בקטריאלי של יחידות משנה הפרוטאזום מאפשר ניתוח מהיר אבל לא יכול לחשוף ביניים הרכבה מפתח. אנו מציעים שילוב Coexpression עם lysate ער...

מתחמי multiprotein לבצע פעילות הסלולר קריטית רבה 1. עבור רבי קומפלקסים אלה, הרבה יותר ידוע על המבנה שלהם ותפקוד מאשר על ההרכבה שלהם 2,3. הפרוטאזום הוא מורכב אחד כזה והוא נמצא בכל תחומי החיים. אאוקריוטים, מכונה מולקולרית זה הוא הליבה של המערכת הפרוטסומית / יוביקוויטין (UPS) ומספק את המסלול העיקרי של פירוק חלבונים תאיים 4. הפרוטאזום איקריוטיים (המכונה הפרוטאזום 26s) מורכב משני מכלולים תת מרכזיים: א הפרוטאזום 20S, או חלקיקי Core (CP) 5, שניתן כתרים על קצוות אחד או שניהם על ידי חלקיק 19S תקין (RP) 6. הפרוטאזום 20S הוא פרוטאז מידור גדול. המבנה הרביעון שלה נשמר לחלוטין בכל תחומי חיים והוא מורכב ערימה של ארבע טבעות שבע membered המכיל שני סוגים של יחידות משנה הקשורות מבני, α; ובטא 5,7,8. אאוקריוטים, שתי הטבעות החיצוניות כל מונה שבע יחידות משנת α ברורות ואת שתי הטבעות פנימיות כל מונה שבע יחידות משנת β ברורות; פעילות פרוטאוליטים מתגוררת בתוך שלוש של יחידות מהשנה β. לעומת זאת, טבעות CP של קדומים וחיידקים הן בדרך כלל מורכבות רק סוג אחד של α וסוג אחד של למקטע β. פרוטאזומים Archaeal סיפקו מערכת מודל חשוב ללמוד הרכבה הפרוטאזום הן בשל פשטות ההלחנה שלהם ושיתוף שלהם מנגנון הרכבה משותף עם עמיתיהם איקריוטיים שלהם 9-13. בקיצור, יחידות משנה α להרכיב לטבעות α ראשונה, אשר לשמש פיגום שעליו בטא יחידות משנה להרכיב. וכתוצאה מכך חצי פרוטאזומים (α 7 β 7) dimerize, והוליד התאספו מלא CP (α 7 β 7 β 7 α 7). במהלך dimerization, את propeptides להציג על יחידות משנה בטאיוסרו autocatalytically, חשיפת threonines N-terminal קטליטי. השימוש פרוטאזומים archaeal מודל הרכבה לעתים קרובות מנצל את הייצור של חלבונים הפרוטאזום archaeal רקומביננטי coli Escherichia. זוהי גישה כדאית משום שהוא מאפשר יחידות משנה להיות מיוצר בצירופים שונים, כמו גם WT וגרסאות מוטציה, בתוך הפונדקאי כי אינו מייצר פרוטאזומים משלה. ניטור ההרכבה של קומפלקסי חלבונים רבים ביוכימית דורש סוג כלשהו של שיטת חלוקת המפריד מתחמים מורכבים במלואו מן ביניים ומבשרי הרכבה. בשל יכולת בפתרון מעולה, nondenaturing polyacrylamide ג&#39;ל אלקטרופורזה (עמוד) הוכיח להיות שימושי במיוחד החלוקה של מתחמי multiprotein גדולים השונים 14-17. השילוב של ייצור הפרוטאזום archaeal רקומביננטי עמוד nondenaturing הפכה גישה חזקה ב dissectinהפרוטאזום גרם הרכבת 9,11,12,18. עם זאת, השיטה הרגילה על ידי איזו גישה זו מיושמת (כלומר. דרך Coexpression רקומביננטי של יחידות משנת α ו β) יש חסרון חשוב. תגובות עצרת מאורגנות על בסיס שיתופיות וריכוז-תלוי מאוד 3. בהתחשב בכך את ריכוז החלבון בתוך התאים הוא גבוה מאוד 19, בשל תופעות נפח נשלל, תגובות הרכבה להמשיך במהירות in vivo. מכאן אפשר לפספס ביניים הרכבה חשובים כאשר יחידות משנת α ו β הן coexpressed. כאן, אנו טוענים בגישה המשולבת במחקר של הרכבה הפרוטאזום באמצעות יחידות משנה הפרוטאזום archaeal רקומביננטי. לפי גישה זו, הן שיטות ערבוב Coexpression ו lysate מועסקים. הראשון מאפשר ניתוח מהיר של הרכבה כי Coexpression הוא פחות עבודה אינטנסיבית. זה האחרון תלוי ביטוי נפרד של תת-יחידות α ו β, ואחריו ערבוב. למרות requi זהמיל קצת יותר מאמץ מאשר Coexpression, זה בהחלט פיצו על ידי היכולת לזהות ביניים כי הם הפסידו במהלך Coexpression. יחד, שתי שיטות יכולות לספק תמונה שלמה יותר של הרכבה הפרוטאזום.

<table><tbody><tr><td>Acrylamide (40%) solution</td><td>Biorad</td><td>1610104</td><td>Unpolymerized acrylamide is a neurotoxin. Wear proper protective gear</td></tr><tr><td>Amicon ultra 0.5 mL centrifugal filters</td><td>EMDMillipore</td><td>UFC501024</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Sigma</td><td>A3678</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A7699</td><td></td></tr><tr><td>BCA assay kit</td><td>Pierce</td><td>23225</td><td></td></tr><tr><td>Bisacrylamide (2%) solution</td><td>Biorad</td><td>1610142</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>B8026</td><td></td></tr><tr><td>DNaseI</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Thermo Fisher</td><td>BP172</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21 competent cells</td><td>EMD Millipore</td><td>69450</td><td></td></tr><tr><td>GelCode Blue</td><td>Thermo Fisher</td><td>24592</td><td>Colloidal coomassie stain reagent for gels</td></tr><tr><td>Gel doc EZ system</td><td>Biorad</td><td>1708270</td><td>Gel documentation system</td></tr><tr><td>Gel releasers</td><td>Biorad</td><td>1653320</td><td>Wedge shaped plastic used to separate gel plates; useful for spreading liquid.</td></tr><tr><td>Glass rod</td><td>Thermo Fisher</td><td>11-380B</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>49767</td><td></td></tr><tr><td>Glycine</td><td>Thermo Fisher</td><td>BP3865</td><td></td></tr><tr><td>Hamilton syringe</td><td>Thermo Fisher</td><td>14-813-38</td><td>Glass syringe for loading gels</td></tr><tr><td>HEPES</td><td>US Biologicals</td><td>H2010</td><td></td></tr><tr><td>HMW Native calibration kit</td><td>GE Healthcare</td><td>170445-01</td><td>High molecular weight protein standards</td></tr><tr><td>Hoefer SG30</td><td>Thermo Fisher</td><td>03-500-277</td><td>Gradient maker</td></tr><tr><td>Imidazole</td><td>US Biologicals</td><td>280671</td><td></td></tr><tr><td>IPTG</td><td>US Biologicals</td><td>I8500</td><td>For induction of protein expression</td></tr><tr><td>Isopropanol</td><td>Thermo Fisher</td><td>BP26181</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>US Biologicals</td><td>K0010</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma</td><td>L6876</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fluka analytical</td><td>630680</td><td></td></tr><tr><td>Mini Protean Tetra Cell</td><td>Biorad</td><td>1658002EDU</td><td>Gel electrophoresis apparatus</td></tr><tr><td>NaCl</td><td>Thermo Fisher</td><td>S640-3</td><td></td></tr><tr><td>NaOH</td><td>Thermo Fisher</td><td>S318-1</td><td></td></tr><tr><td>Pefabloc SC</td><td>Roche</td><td>11429876001</td><td>Protease inhibitor</td></tr><tr><td>pET42</td><td>EMD Millipore</td><td>70562</td><td>Expression plasmid</td></tr><tr><td>Precision plus all blue standard</td><td>Biorad</td><td>1610373</td><td>Molecular protein standard for SDS-PAGE</td></tr><tr><td>Quickchange mutagenesis kit</td><td>Agilent technologies</td><td>200521</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Thermo Fisher</td><td>BP166</td><td></td></tr><tr><td>Suc-LLVY-AMC</td><td>Enzo lifesciences</td><td>BML P802-0005</td><td>Fluorogenic substrate</td></tr><tr><td>Talon Metal Affinity Resin</td><td>Clontech</td><td>635502</td><td>Immobilized Cobalt Affinity Resin</td></tr><tr><td>TEMED</td><td>Sigma</td><td>T7024</td><td></td></tr><tr><td>Tris</td><td>US Biologicals</td><td>T8600</td><td></td></tr><tr><td>Triton-X100</td><td>Sigma</td><td>93426</td><td></td></tr><tr><td>Tryptone</td><td>Bacto BD</td><td>211699</td><td></td></tr><tr><td>UV sample tray</td><td>Biorad</td><td>1708271</td><td>For UV imaging of gels</td></tr><tr><td>Yeast extract</td><td>Bacto BD</td><td>212720</td><td></td></tr></tbody></table>

examining proteasome assembly with recombinant archaeal proteasomes and nondenaturing page:  the case for a combined approach

הפרוטאזומים נמצאים בכל תחומי החיים. הם מספקים את המסלול העיקרי של פירוק חלבונים תאיים אאוקריוטים, אם כי הרכבה שלהם אינה מובנת לחלוטין. כל פרוטאזומים מכילים חלקיק הליבה נשמרת מבנית (CP), או הפרוטאזום 20S, המכיל שתי טבעות למקטע β heptameric דחוקה בין שתי טבעות למקטע α heptameric. פרוטאזומים 20S Archaeal פשוטים לעומת בעלי הרכב לעמיתיהם איקריוטיים שלהם, בעודם חולקים מנגנון הרכבה משותף. כתוצאה מכך, פרוטאזומים 20S archaeal להמשיך להיות מודל חשוב להרכבת הפרוטאזום איקריוטיים. באופן ספציפי, ביטוי רקומביננטי של פרוטאזומים 20S archaeal בשילוב עם ג&#39;ל אלקטרופורזה polyacrylamide nondenaturing (עמוד) הניב תובנות חשובות רבות לתוך biogenesis הפרוטאזום. כאן, אנו דנים אמצעי לשפר את האסטרטגיה הרגילה של Coexpression של α הפרוטאזום archaeal ובטא יחידות משנה לפני nondenaturing עמוד. אנו מראים כי למרות מהירים ויעילים, גישת Coexpression לבדו יכולה לפספס ביניים הרכבת מפתח. במקרה של הפרוטאזום, Coexpression לא יכול לאפשר זיהוי של-הפרוטאזום וחצי, טבעת-α מלאה המכילה ביניים אחת β-טבעת אחד שלם. עם זאת, ביניים זו מזוהה בקלות באמצעות ערבוב lysate. אנו מציעים שילוב Coexpression עם ערבוב lysate מניב גישה יותר יסודית בניתוח הרכבה, עדיין נותר העבודה nonintensive. גישה זו עשויה להיות שימושית לחקר מתחמי multiprotein רקומביננטי אחרים.

הפרוטאזום הרכבה (איור 1) מתחילה כאשר יחידות משנת אלפא לשלב טבעות טופס 9. אפשר להדגים זאת כאשר יחידות משנה אלפא מן archaeon Methanococcus maripaludis S2 מבוטאים החיידק כמו C- סופני hexahistidine מתויג (מתויג שלו) נגזרים (טבלה 1). כאשר α-שלו רקומביננטי החלבון היה ?...

Watch this Scientific Journal Video about Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach at JoVE.com

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

פרוטוקול זה משתמש בשני Coexpression למקטע ו למקטע postlysis ערבוב לבדיקה יסודית יותר של הרכבת הפרוטאזום רקומביננטי.

בחינת העצרת הפרוטאזום עם רקומביננטי Archaeal הפרוטאזומים ועמוד Nondenaturing: The Case for גישה משולבת

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

examining-proteasome-assembly-with-recombinant-archaeal-proteasomes

Research

JoVE Journal

Biochemistry

6.6K Views.  Indiana University-Purdue University, Indianapolis (IUPUI).  פרוטוקול זה משתמש בשני Coexpression למקטע ו למקטע postlysis ערבוב לבדיקה יסודית יותר של הרכבת הפרוטאזום רקומביננטי. 

Video: Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

אפיון של קומפלקסים חלבונים Multi-למקטע של MxA האדם שימוש ללא denaturing polyacrylamide ג&#39;ל אלקטרופורזה

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

Multimer-עמוד: שיטה לכידת וחלבון פתרון מתחמי דגימות ביולוגיות

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

פתרון מתחמי פרוטאין טהור זיקה לפי דף Native הכחול פרופיל מתאם חלבון

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

השימוש פיוז'ן רקומביננטי חלבונים פלטפורמה Assay פרוטאז פלורסנט, Renaturation שלהם בג'ל

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

אלקטרופורזה היברידית ברורה/כחולה עבור הפרדה וניתוח של שרשרת נשימה מיטוכונדריאלי מערכות-העל

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

ג&#39;ל כחול Native polyacrylamide אלקטרופורזה (BN-PAGE) עבור ניתוח של מתחמי multiprotein מ lysates נייד

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Biology

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

מנסה לאמוד השפלה proteasomal במערכת תא ללא בצמחים

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

מבחנים עבור הפירוק של חלבונים בתאים Misfolded

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

שילוב cross-linking כימי ספקטרומטר מסה של מתחמי חלבון שלם ללמוד את הארכיטקטורה של יחידת משנה רב חלבון להרכבות

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing conditions could be achieved in different plant species. Native protein:protein and protein:nucleic acid complexes from phloem samples have as yet scarcely been analyzed, although they might play important roles in maintenance of this specialized compartment or in long-distance signaling. Large molecular assemblies can be isolated using a blue native gel electrophoresis (BN-PAGE). Their protein components can be separated by a subsequent sodium dodecyl sulfate PAGE (SDS-PAGE). However, proteins with similar molecular weights co-migrate, what can hinder protein identification by mass spectrometry. Combining BN-PAGE with two different denaturing gel electrophoresis steps, namely Tris-Tricine-urea and SDS-PAGE, enables the additional separation of proteins according to their hydrophilicity/hydrophobicity and thus increases resolution and the success of protein identification. It even allows distinguishing proteins that only differ in their posttranslational modifications. In addition, blue native northern blotting can be applied to identify the RNA components in macromolecular complexes. We show that our protocol is suitable to unravel the protein and RNA components of native protein:protein and ribonucleoprotein (RNP) complexes occurring in phloem samples. Combining a blue native PAGE with two different denaturing PAGE steps can help to separate different kinds of large protein complexes, and also enables an increased identification rate of their components by mass spectrometry. Furthermore, the protocol is robust enough to simultaneously detect potentially bound nucleic acids within single protein complexes.

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.

שיפה Sap דגימה של כרוב napus עבור תלת-ממד-דף של חלבונים, קומפלקסים Ribonucleoprotein

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing ...

בחינת העצרת הפרוטאזום עם רקומביננטי Archaeal הפרוטאזומים ועמוד Nondenaturing: The Case for גישה משולבת

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אפיון של קומפלקסים חלבונים Multi-למקטע של MxA האדם שימוש ללא denaturing polyacrylamide ג'ל אלקטרופורזה