This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

Procedure sperimentali con i topi sono stati approvati dal Comitato di McGill University (protocollo 4966) Animal Care. 1. Preparazione di estratti proteici di cellule in coltura <ol><li> Lavare le cellule in coltura per due volte con 10 ml di fosfato ghiacciata salina tamponata (PBS). </li><li> Raschiare cellule aderenti sia con un poliziotto gomma o un raschietto cellulare plastica in 1 ml di sospensione di PBS, trasferimento fredda in una provetta da 1,5 ml microcentrifuga. </li><li> Spin in una microcentrifuga per 5 minuti a 700 xg, a 4 ° C. Aspirare PBS. </li><li> Aggiungere 100 ml di tampone di lisi citoplasmatica ghiacciata (Tabella 1) per 10 7 cellule, e pipetta su e giù. </li><li> Incubare in ghiaccio per 20 min. </li><li> Spin per 10 min a piena velocità in una microcentrifuga a 4 ° C. </li><li> Pellet Rifiuta. Trasferire il surnatante in una nuova provetta da 1,5 ml microcentrifuga e tenere in ghiaccio. </li><li> Detconcentrazione ermine di proteine ​​(di solito 1-10 mg / mL) con il saggio Bradford 23. </li><li> Aliquota e cellulari negozio estratti a -80 ° C fino al momento dell&#39;uso. </li></ol> 2. Preparazione di estratti di proteine ​​di topo fegato e milza <ol><li> Euthanize un mouse con CO 2 inalazione. </li><li> Posare l&#39;animale eutanasia su un tappetino pulito su una tavola di dissezione. Aprire l&#39;addome con le forbici. </li><li> Staccare il fegato e la milza, utilizzando forbici e pinze, e lavare ogni tessuto in circa 50 ml ghiacciata PBS. </li><li> Subito tagliare tessuti in piccoli pezzi con un bisturi (ad esempio: circa 1 - 2 mm 3). </li><li> Senza indugio, mettere pezzi di tessuto in una fresca cryotube e quindi far scattare congelarli in azoto liquido. Conservare snap-congelati aliquote tessuto a -80 ° C fino al momento dell&#39;uso. </li><li> Omogeneizzare un pezzo di tessuto congelato (circa 1 - 2 mm 3) in 0,25-0,5 ml dighiacciato tampone di lisi citoplasmatica (Tabella 1) con un omogeneizzatore tessuto per 10 sec. </li><li> Trasferimento omogeneizzato a 1,5 ml provetta e freddo in ghiaccio per 20 min. </li><li> Spin per 10 min a piena velocità in una microcentrifuga a 4 ° C. </li><li> Eliminare pellet e trasferimento surnatante in nuova provetta da 1,5 ml microcentrifuga. Mantenere su ghiaccio. </li><li> Determinare la concentrazione di proteine ​​(di solito 1-10 mg / mL) con il saggio Bradford 23. </li><li> Aliquota e cellulari negozio estratti a -80 ° C fino al momento dell&#39;uso. </li></ol> 3. Preparazione di marcata radioattivamente IRE-probe <ol><li> Linearizzare il IRE contenenti plasmide I-12.CAT 22 incubando a 37 ° C per 1 ora con l&#39;endonucleasi di restrizione XbaI (1 U per pg di plasmide), che scinde a valle della sequenza IRE. Il plasmide linearizzato sarà utilizzato come stampo per la trascrizione in vitro. </li><li> Impostare unn in vitro reazione di trascrizione in un volume totale di 20 microlitri. Utilizzare le soluzioni azionari riportati in Tabella 2 e aggiungere: 1 ml modello plasmide linearizzato, buffer di trascrizione 4 ml; 1 ml mix di ATP mix / CTP / GTP, 10 ml [α- 32 P] -UTP, 2 microlitri ditiotreitolo, inibitore RNasi 1 ml e 1 ml T7 RNA polimerasi. Mescolare pipettando su e giù. </li><li> Incubare a 40 ° C per 1 ora 24. </li></ol> 4. Purificazione di marcata radioattivamente IRE-probe <ol><li> Terminare la reazione di trascrizione in vitro con l&#39;aggiunta di 1 ml di 0,5 M EDTA, pH 8. Mix pipettando su e giù. </li><li> Aggiungere 10 ml di 10 mg / ml tRNA, come supporto per una migliore precipitazione. Mescolare pipettando su e giù. </li><li> Aggiungere 82,5 ml di 3 M acetato di ammonio. Mescolare nel vortex. </li><li> Aggiungere 273 ml di etanolo. Mescolare nel vortex. </li><li> Lasciar riposare a temperatura ambiente per 5 min. </li><li> Spin per 10 min a piena velocità in una microcentrifuga a RT. Surnatante Rifiuta. </li><li> Lavare pellet con 100 ml di etanolo al 70%. </li><li> Spin per 10 min a piena velocità in una microcentrifuga a RT. Surnatante Rifiuta. </li><li> Aria pellet secco per 10 min. </li><li> Risospendere pellet in 100 ml di bidistillata, precedentemente sterilizzato in autoclave H 2 O. </li><li> Quantificare la radioattività in un contatore a scintillazione liquida, sonda IRE aliquota radiomarcato e conservare a -80 ° C fino al momento dell&#39;uso. Aliquote congelate possono essere usati fino a 3 settimane. </li></ol> 5. Preparazione di un gel di poliacrilammide nativo per EMSA <ol><li> Montare il gel (16 x 16 cm), utilizzando 1,5 millimetri distanziali e pettine. </li><li> Per preparare un gel di poliacrilammide nativo il 6%, utilizzare le soluzioni madre indicate nella Tabella 3 Mescolare 7,5 ml di 40% acrilamide:. Bisacrilammide, 5 ml di 5x TBE e 37,5 mldoppia H distillata 2 O. </li><li> Aggiungere 0,5 ml di 10% persolfato preparata ammonio (APS) e 25 ml tetrametiletilendiammina (TEMED). </li><li> Versare immediatamente la soluzione acrilamide al gel e lasciare polimerizzare. Attendere per circa 30 min. </li><li> Montare l&#39;apparecchiatura elettroforesi con il gel, riempire i serbatoi con 0,5x TBE e connettersi con l&#39;alimentazione. </li></ol> 6. Electrophoretic Mobility Shift Assay <ol><li> Diluire 25 mg di estratto proteico da cellule o tessuti con citoplasmatica tampone di lisi (Tabella 1) in un volume totale di 10 microlitri in (concentrazioni proteiche inferiori possono anche essere utilizzati). Mantenere su ghiaccio. Se necessario, aggiungere 1 ml di 1: 4 diluito 2-mercaptoetanolo (2-ME) per attivare IRP1 dormiente (concentrazione finale: 2%) 25. </li><li> Diluire la sonda radioattivo IRE in doppia H 2 O distillata a 200.000 cpm / ml, denaturare calore a 95 °C per 1 min e raffreddare a temperatura ambiente per almeno 5 minuti. </li><li> Impostare una reazione EMSA aggiungendo 1 ml di sonda IRE radioattivo al estratto proteico. </li><li> Incubare per 20 minuti a RT. </li><li> Aggiungere 1 ml di 50 mg / ml di eparina alla reazione (per inibire le interazioni proteina non specifiche con la sonda 9) e continuare l&#39;incubazione per altri 10 min. Aliquota della soluzione madre di eparina (50 mg / ml) e conservare a -80 ° C. </li><li> Quando si utilizzano sonde lunghe radiomarcati (&gt; 60 nucleotidi), aggiungere 1 ml RNasi T1 (1 U / ml) e incubare per 10 minuti a RT per ridurre proteina legame non specifico alla sonda, e per consentire una migliore separazione della RNA / proteine complesso durante l&#39;elettroforesi. </li><li> Aggiungere 3 ml di tampone di caricamento (80% glicerolo + bromofenolo blu), mescolare e carico sul non-denaturazione gel di poliacrilammide al 6%. </li><li> Eseguire il gel per 60 min a 130 V (5 V / cm). </li><li> Trasferimento il gel su una grande carta da filtro e asciutta. </li><li> Esporre a un film e sviluppare autoradiografia. Tempo di esposizione può variare da 1 ora (o meno) fino a O / N. </li></ol>

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The authors declare that they have no competing financial interests.

Qui, descriviamo un protocollo che è stato sviluppato per studiare le attività IRE vincolanti di IRP1 e IRP2, e mostriamo dati rappresentativi. Utilizzando diverse sonde, questo protocollo può essere regolato anche per lo studio di altre proteine-RNA vincolante. Un punto critico è la dimensione della sonda. L&#39;utilizzo di sonde lunghe, che è comune quando l&#39;esatto sito di legame è sconosciuto, può portare a complessi RNA / proteine ​​che non migrano in modo diverso rispetto alla RNA gratuito. In questo...

L&#39;EMSA è stato originariamente sviluppato per studiare l&#39;associazione di proteine ​​che legano il DNA con sequenze di DNA bersaglio 1,2. Il principio è simile per le interazioni RNA / proteine ​​3, che è il focus di questo articolo. Brevemente, l&#39;RNA è caricato negativamente e migrerà verso l&#39;anodo durante non denaturante elettroforesi in poliacrilammide (o agarosio gel). Migrazione nel gel dipende dalla dimensione del RNA, che è proporzionale alla sua carica. Legame specifico di una proteina di RNA altera la sua mobilità, e le migra complessi più lento rispetto alla RNA libera. Ciò è dovuto principalmente ad un aumento della massa molecolare, ma anche ad alterazioni nella carica e possibilmente conformazione. Utilizzando un RNA marcato come sonda permette un facile monitoraggio del &quot;ritardo gel&quot; o &quot;bandshift&quot;. L&#39;utilizzo di 32 sonde RNA P-marcato è molto comune e offre alta sensibilità. La migrazione di RNA / complessi proteici e RNA libero vengono rilevatimediante autoradiografia. Inconvenienti sono breve emivita di 32 P (14,29 giorni), il progressivo deterioramento della qualità della sonda a causa radiolisi, il requisito di una licenza radioattività e infrastruttura per lavoro radioattività, e potenziali problemi di biosicurezza. Pertanto, sono stati sviluppati metodi non isotopici alternativi per etichettare la sonda RNA, per esempio con fluorofori o biotina, che consentono il rilevamento da parte di imaging fluorescente o chemiluminescente 4,5. Limitazioni di questi metodi sono il costo elevato e spesso ridotta sensibilità rispetto all&#39;etichettatura isotopica, e il potenziale di etichette non isotopici di interferire con l&#39;interazione RNA / proteine. Gel non denaturante di poliacrilammide sono adatti per la maggior parte delle applicazioni di EMSA e sono comunemente usati. Occasionalmente, gel di agarosio possono rappresentare un&#39;alternativa per l&#39;analisi di grandi complessi. Il principale vantaggio di EMSA è che combina semplicità, sensibilità, robustezza e 4 &lt;/ Sup&gt;. Il test può essere completato nel giro di poche ore e non richiede una strumentazione sofisticata. Interazioni RNA / proteine ​​possono essere rilevati da EMSA a concentrazioni basse come 0,1 nm o inferiori, e all&#39;interno di una vasta gamma di condizioni vincolanti (pH 4,0-9,5, la concentrazione di sale monovalente 1-300 mM, e la temperatura 0 - 60 ° C). RNA / proteine ​​formazione del complesso può essere studiato mediante il saggio filtro vincolante. Questa è una procedura semplice, veloce ed economico basato sulla conservazione dei complessi RNA / proteine ​​in un filtro di nitrocellulosa, mentre una sonda di RNA libera attraversa 6. Rispetto al EMSA, è limitato nei casi in cui la sonda RNA contiene più siti di legame, o l&#39;estratto grezzo contiene più di un RNA-binding proteins che si legano alla sonda nello stesso sito. Mentre molteplici interazioni RNA / proteine ​​sfuggiranno rilevamento da parte del saggio di filtro di legame, possono essere facilmente visualizzati tramite EMSA. In alcuni casi, la visualizzazione è vigilian possibile quando due complessi RNA / proteine ​​co-migrazione (per esempio, IRP1 umana / IRE e IRP2 / complessi IRE), con l&#39;aggiunta di un anticorpo contro una delle RNA-binding proteins alla reazione EMSA, ottenendo un&#39;ulteriore ritardo sul gel ( &quot;supershift&quot;) 7. L&#39;EMSA è stato ampiamente utilizzato per studiare IRP1 e IRP2, che sono regolatori post-trascrizionali del metabolismo del ferro 8-10. Operano legandosi a IRE, strutture tornante filogeneticamente conservate nelle UTR di diversi mRNA 11. IRE sono stati scoperti nel mRNA codificanti ferritina 12 e transferrina recettore 1 (TfR1) 13, proteine ​​di stoccaggio ferro e l&#39;assorbimento, rispettivamente. Più tardi, IRE sono stati trovati in specifici eritroide aminolevulinato sintasi (ALAS2) 14, aconitasi mitocondriale 15, ferroportina 16, divalente metallo transporter 1 (DMT1) 17, ipossia fattore inducibile 2 <em&gt; α (HIF2α) 18, e altri mRNA 19-21. Il H- prototipi e mRNA L-ferritina contengono una IRE nella loro 5 &#39;UTR, mentre TfR1 mRNA contiene più IRE nel suo 3&#39; UTR. Interazioni IRE / GIV specificamente inibiscono ferritina traduzione mRNA da stericamente bloccando la sua associazione delle subunità ribosomiale 43S; inoltre, si stabilizzano TfR1 mRNA contro taglio endonucleolitico. IRP1 e quota IRP2 ampia similarità di sequenza e presentano un&#39;elevata attività-IRE legame in cellule di ferro-fame. Nelle cellule ferro-piena, IRP1 assembla un cubano Fe-S cluster che converte in aconitasi citosolica a scapito della sua attività IRE vincolante, mentre IRP2 subisce la degradazione del proteasoma. Pertanto, le interazioni IRE / IRP dipendono dal livello di ferro cellulare, ma sono anche regolati da altri segnali, quali H 2 O 2, ossido nitrico (NO) o ipossia. Qui, descriviamo il protocollo per valutare l&#39;attività IRE vincolante da estratti di cellule e tessuti greggi da EMSA. Abbiamo utilizzato un 32 P-marcato H-ferritina sonda IRE che è stato generato mediante trascrizione in vitro da un modello DNA plasmidico (I-12.CAT), dove la sequenza IRE è stato inizialmente introdotto in orientamento senso valle del sito polimerasi T7 RNA da clonazione di ricotto oligonucleotidi sintetici 22.

<table border="0" cellpadding="0" cellspacing="0" width="430">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:195px;">Name of the Materials&#160;</td>
			<td style="width:119px;">Company</td>
			<td style="width:117px;">Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">leupeptin</td>
			<td>SIGMA</td>
			<td>L2884</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PMSF</td>
			<td>SIGMA</td>
			<td align="right">78830</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BioRad Protein Assay</td>
			<td>BIORAD</td>
			<td>500-0006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T7 RNA polymerase</td>
			<td>Thermoscientific</td>
			<td>EPO111</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase Inhibitor</td>
			<td>Invitrogen</td>
			<td>15518-012</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">UTP [alpha-32P]</td>
			<td>Perkin-Elmer</td>
			<td>NEG507H</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation liquid</td>
			<td>Beckman Coulter</td>
			<td align="right">141349</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean II xi Cell&#160;&#160;</td>
			<td>BIORAD</td>
			<td>165-1834</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
		</tr>
	</tbody>
</table>

electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

Interazioni RNA / proteine ​​sono fondamentali per vie di regolazione post-trascrizionale. Tra le RNA-binding proteins citosoliche più caratterizzati sono proteine ​​regolatrici del ferro, IRP1 e IRP2. Si legano al ferro elementi responsivi (IRES) all&#39;interno delle regioni non tradotte (UTR) dei diversi mRNA bersaglio, controllando così la traduzione mRNA o la stabilità. Interazioni IRE / IRP sono stati ampiamente studiati da EMSA. Qui, descriviamo il protocollo EMSA per analizzare l&#39;attività IRE vincolante di IRP1 e IRP2, che può essere generalizzato per valutare l&#39;attività di altri RNA-binding proteins pure. Un lisato proteina grezza contenente una proteina legante l&#39;RNA, o una preparazione purificata di questa proteina, viene incubato con un eccesso di 32 sonda RNA P-marcato, consentendo la formazione del complesso. L&#39;eparina è aggiunto precludere proteina non specifica per sondare vincolante. Successivamente, la miscela viene analizzata mediante elettroforesi non denaturante su un gel di poliacrilammide. La sonda gratuitomigra veloce, mentre l&#39;RNA / proteine ​​mostre complesse mobilità ritardato; di conseguenza, la procedura è chiamata anche &quot;il ritardo gel&quot; e il dosaggio &quot;bandshift&quot;. Dopo il completamento della elettroforesi, il gel viene essiccato e complessi RNA / proteine, così come sonda free, vengono rilevati mediante autoradiografia. L&#39;obiettivo generale del protocollo è quello di individuare e quantificare IRE / IRP e altre interazioni RNA / proteine. Inoltre, EMSA può anche essere usato per determinare la specificità, l&#39;affinità di legame, e stechiometria dell&#39;interazione RNA / proteina in esame.

Una sonda radiomarcata IRE è stato preparato, come descritto nelle sezioni 3 e 4 del protocollo. La sequenza della sonda era 5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUG C UUCAA C AGUGC UUGGA CGGAUCCU-3 &#39;; i nucleotidi in grassetto rappresentano un residuo C spaiato e il ciclo, che sono caratteristiche critiche IRE. La radioattività specifica della sonda era 4,5 x 10 9 cpm / mg di RNA. Per valutare gli effetti delle perturbazioni ferro sull&#...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Qui vi presentiamo un protocollo per analizzare le interazioni RNA / proteine. Il saggio spostamento mobilità elettroforetica (EMSA) si basa sulla migrazione differenziale di complessi RNA / proteine ​​e RNA libero durante elettroforesi su gel nativo. Utilizzando una sonda RNA radioattivo, RNA complessi / proteici possono essere visualizzati mediante autoradiografia.

Electrophoretic Maiusc Mobility Assay (EMSA) per lo studio delle interazioni RNA-proteina: La IRE / IRP Esempio

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.2K Views.  Jewish General Hospital.  Qui vi presentiamo un protocollo per analizzare le interazioni RNA / proteine. Il saggio spostamento mobilità elettroforetica (EMSA) si basa sulla migrazione differenziale di complessi RNA / proteine ​​e RNA libero durante elettroforesi su gel nativo. Utilizzando una sonda RNA radioattivo, RNA complessi / proteici possono essere visualizzati mediante autoradiografia. 

Video: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Ricerca

Biologia

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Ottimizzato fibrina Gel Bead Assay per lo studio della angiogenesi

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated modello animale Stimoli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositivo per il recupero di campioni crioconservati cadere accidentalmente nelle cisterne criogeniche

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Un liquido fase di cattura test affinità utilizzando biglie magnetiche per studiare interazioni proteina-proteina: il poliovirus-Nanobody Esempio

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Il MultiBac Protein Complex Piattaforma di produzione al EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Germogliamento lievito proteine ​​estrazione e purificazione per lo Studio della funzione, interazioni e modificazioni post-traduzionali

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando singola molecola di manipolazione e di imaging per lo studio delle interazioni proteina-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Screening per Funzionale non codificante Varianti genetiche Utilizzando Electrophoretic Mobility Shift Assay (EMSA) e DNA-affinità precipitazioni Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...