עבודה במעבדה של המחברים נתמכת על ידי מענק מהמכון הלאומי למדעי רפואה כלליים (GM41628) ועל ידי מענק למכון Stowers למחקר רפואי מהלן נלסון רפואי קרן המחקר בקרן קהילת קנזס רבתי העיר.

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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

מחקרים מבניים ותפקודיים של רב למקטע מתחמי שיפוץ הכרומטין יונקים מאאוקריוטים גבוהים כבר הקשו על ידי הקושי של הכנה ביוכימית כמויות שימושיות של מתחמים כגון המכילים יחידות משנה שעברו מוטציה או חסרי יחידות משנה מסוימות לגמרי. ישנם מספר של מכשולים טכניים: ראשית, מניפולצי...

מתחמי שיפוץ הכרומטין משפחת SNF2 אבולוציונית שמורים הם רגולטורים מרכזיים של ארגון הכרומטין ונגישות DNA 1. מתחמי שיפוץ אלה תמיד כוללים מקטע מרכזי כמו SNF2-ATPase, אשר, במקרים מסוימים, מרכיב בחלבוני אבזר שונים ויוצר מכלולי מקרו מולקולרי רב למקטע. כדי לחקור את הפרטים המולקולריים של תהליך שיפוץ הכרומטין תלוי ATP, זה חשוב להבין את תרומתם של תת קבוצות מסוימות של יחידות משנה ו / או מבני תחום לפעילות של מתחמים. ניתוח מסוג זה דורש את היכולת ליצור מתחמי מוטציה נקיים במיוחד שחסרות יחידות משנה חלבון מסוימות או מבני תחום. מחקרי מבנה פונקציה קודמים של מתחמי שיפוץ הכרומטין תלוי ATP התמקדו באופן נרחב במערכת שמרי המודל בשל manipulability מעולה של הגנום השמרים (ראה, למשל, שופטים 1-4). בהתחשב בשימורהרכב למקטע ופונקציונליות בין מתחמי שיפוץ orthologous, מחקרים של המבנה והתפקוד של קומפלקסי שיפוץ שמרים סיפקו תובנות חשובות עמיתיהם באאוקריוטים גבוהים יותר. עם זאת, מינים ספציפיים ניכרים הבדלים בין מתחמי שיפוץ קיימים, כתוצאה מרווח או הפסד של מינים ספציפיים יחידות משנה, הרווח או הפסד של תחומים מינים ספציפיים של יחידות משנה נשמרו, והשתנות רצף בתוך תחומים משומרים של יחידות משנה נשמרת. יכולים באופן עקרוני להיות מונעים את ההבדלים כאמור על ידי הצורך בתאים האיקריוטים גבוהים יותר להסתגל לסביבות מולקולריות ותאיות חדשות. לפיכך, ההבנה כיצד יחידות משנה של מתחמי שיפוץ אוקריוטים גבוהים לתרום לתהליך שיפוץ הנוקלאוזום היא בעל ערך, כי זה לא רק גם שופך אור על מנגנונים בסיסיים של תהליך שיפוץ הכרומטין תלוי ATP, אבל יכול לספק תובנה חשובה המנגנונים שבאמצעותם מבנה הכרומטין וביטוי גנים בפואאוקריוטים מוסדרים. עד כה, יש כבר מוגבל רק מחקרים מבניים ותפקודיים של מתחמי שיפוץ הכרומטין יונקים רב למקטע, נובע בחלקו בקשיים בהשגת מתחמים מוגדרים ביוכימית הכרומטין שיפוץ וsubcomplexes. באופן חלקי יש לנו לעקוף את הקשיים הללו לנהלים שיפורטו להלן, שבו לטיהור immunoaffinity משמשת להכנת subcomplexes INO80 או INO80 שלם מהתאים אנושיים ביציבות להביע epitope דגל N-סופני מתויג סוג בר או גרסאות מוטציה של Ino80 5-7 (איור 1) . כדי להשיג מתחמי INO80 שלמים מהתאים אנושיים, רקומבינציה בתיווך FLP משמשת ליצירת שורות תאי HEK293 מהונדסים ביציבות להביע cDNAs מתויגים epitope דגל קידוד יחידות משנה של המתחם INO80 8-10. בגלל ביטוי יתר של יחידות משנה INO80 יכול להיות רעיל במידה מסוימת, יש צורך לבודד ולשמור על שורות תאים משובטים תחת שיתוף סלקטיביnditions כדי להבטיח ביטוי transgene יציב במהלך הקטעים רבים הנדרשים להרחבה של תרביות תאים בקנה מידה גדולה. כדי להשיג subcomplexes INO80 קטן יותר המכיל רק קבוצת משנה של יחידות משנה, השתמשנו בהצלחה שתי גישות (איור 2 א, ב &#39;). בחלק הראשון, אנו יוצרים שורות תאי HEK293 FLP-In ביציבות להביע גרסאות מוטציה של Ino80 שחסרת ​​תחומים הנדרשים לאינטראקציה עם יחידות משנה ספציפיות 5. לחלופין, מציאה בתיווך siRNA משמשת לרוקן למקטע הרצוי מהתאים לבטא מקטע מתאים מתויג דגל INO80 (נתונים שלא פורסמו). לבסוף, כדי לטהר את מתחמי INO80 האנושיים, כרומטוגרפיה המבוססת agarose FLAG 11 משמשת להעשרת חלק המכיל INO80 מתמציות גרעיניות, ובכך מפחיתה ביעילות את נוכחותם של חלבונים מזהמים cytosolic בשבריר הסופי המכיל subcomplexes INO80 או INO80 המטוהר.

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			<td height="21" style="height:21px;">PCR thermal cycler PTC 200</td>
			<td>MJ Research</td>
			<td style="width:147px;">PTC 200</td>
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			<td height="21" style="height:21px;width:359px;">roller bottle incubator</td>
			<td style="width:212px;">Bellco biotechnology</td>
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			<td height="21" style="height:21px;width:359px;">Immobilon-FL Transfer Membrane 7 x 8.4</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">IPFL07810</td>
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		<tr height="21">
			<td height="21" style="height:21px;">lubricated 1.5ml microcentrifuge tubes&#160;</td>
			<td style="width:212px;">Costar</td>
			<td style="width:147px;">3207</td>
			<td style="width:173px;"></td>
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generation and purification of human ino80 chromatin remodeling complexes and subcomplexes

מתחמי שיפוץ הכרומטין INO80 להסדיר דינמיקת הנוקלאוזום ונגישות DNA על ידי מזרזות שיפוץ הנוקלאוזום תלוי ATP. מתחמי INO80 אדם מורכבים מ14 יחידות משנה חלבון כולל Ino80, ATPase כמו SNF2, המשמש גם כמקטע קטליטי והפיגום להרכבה של המכלולים. פונקציות של יחידות משנה האחרות והמנגנונים שבאמצעותם הם תורמים לפעילות שיפוץ הכרומטין של מתחם INO80 להישאר הבינו היטב, בין שאר בשל האתגר של יצירת מכלולי INO80 בתאים אנושיים או מערכות ביטוי Heterologous. פרוטוקול יופיטר זה מתאר הליך המאפשר טיהור של subcomplexes שיפוץ הכרומטין INO80 האנושי שחסרי מקטע או קבוצת משנה של יחידות משנה. epitope דגל N-סופני מתויג Ino80 cDNA הם הציגו ביציבות לכליה עוברית אנושית (HEK) 293 שורות תאים באמצעות רקומבינציה בתיווך FLP. במקרה שקבוצת משנה של יחידות משנה של מתחם INO80 הוא bדואר שנמחק, אחד מבטא חלבוני Ino80 במקום מוטציה שחסר את הפלטפורמה הנדרשת להרכבה של תת יחידות אלה. באירוע למקטע בודד הוא להיות מדולדל, siRNAs אחד transfects מיקוד למקטע זה לשורת תאי HEK 293 יציבות להביע FLAG tagged Ino80 ATPase. תמציות גרעיניות מוכנות, וimmunoprecipitation FLAG מתבצעת על מנת להעשיר את השברים חלבון המכילים נגזרי Ino80. הקומפוזיציות של subcomplexes INO80 מטוהר לאחר מכן ניתן לנתח באמצעות שיטות כגון immunoblotting, צביעת כסף, וספקטרומטריית מסה. Subcomplexes INO80 וINO80 שנוצר על פי פרוטוקול זה ניתן לנתח נוסף באמצעות מבחני ביוכימיים שונים, אשר מתוארים בפרוטוקול יופיטר הנלווה. השיטות שתוארו כאן יכולות להיות מותאמות ללימודים של התכונות המבניות ותפקודיות של כל שיפוץ הכרומטין רב למקטע של יונקים ומתחמי שינוי.

איור 1 מציג תרשים זרימה המסכם את הנהלים המשמשים לייצור, לטהר, ולאפיין מתחמי הכרומטין INO80 תלוי ATP אנושיים שיפוץ. כפי שמודגם באיורים 2 ו -3, נהלים אלה מאפשרים את הדור של שני subcomplexes INO80 הסוג וINO80 הפראי שאין תת י...

Watch this Scientific Journal Video about Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes at JoVE.com

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

פרוטוקול זה מתאר הליך להפקת וטיהור מסוג בר וגרסאות מוטציה של מתחם שיפוץ הכרומטין INO80 האנושי. Epitope מתויג גרסאות של יחידות משנה INO80 באות לידי ביטוי ביציבות בתאי HEK293, ומתחמים שלמים ומתחמים חסרי מערכות ספציפיות של יחידות משנה הם מטוהרים על ידי כרומטוגרפיה immunoaffinity.

דור וטיהור של מכלולי אדם INO80 הכרומטין שיפוץ וSubcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

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9.8K Views.  Stowers Institute for Medical Research.  פרוטוקול זה מתאר הליך להפקת וטיהור מסוג בר וגרסאות מוטציה של מתחם שיפוץ הכרומטין INO80 האנושי. Epitope מתויג גרסאות של יחידות משנה INO80 באות לידי ביטוי ביציבות בתאי HEK293, ומתחמים שלמים ומתחמים חסרי מערכות ספציפיות של יחידות משנה הם מטוהרים על ידי כ...

Video: Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

טיהור ויזואליזציה של שפעת מכלולים Ribonucleoprotein ויראלי

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

הלבלב האנושי בידוד איילט: חלק ב: טיהור והתרבות של איי האדם

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

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 ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

הדור של האדם CD40 בתאי B המופעל

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

בידוד הכרומטין על ידי טיהור RNA (ציוץ)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

זיהוי של קומפלקסי חלבונים ב<em&gt; חיידקי Escherichia</em&gt; באמצעות טיהור זיקת פפטיד סדרתית בשילוב עם טנדם המוני ספקטרומטריית

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

דור, טיהור, ואפיון של מיני חלבון DISC1 הסלולריים פולשניים

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

דור של תבנית הכרומטין immunoprecipitation איכות גבוהה עבור ה-DNA רצף תפוקה גבוהה (שבב ואילך)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

הפקה וטיהור יעילה של רקומביננטי Murine Kindlin-3 מתאי חרקים לBiophysical לימודים

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

מבחני ביוכימיים לניתוח פעילויות של ATP תלוי הכרומטין שיפוץ אנזימים

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...