Nous tenons à remercier le laboratoire Lee de l&#39;Université du Minnesota pour fournir les échantillons de tissus de la moelle épinière de souris qui ont été utilisés. Ce travail a été soutenu par le ministère de la Défense Programme Cancer de l&#39;ovaire de la recherche (OCRP) de OC093424 à MB, par le Fonds communautaire de recherche et cancer Randy Shaver MB et par le Minnesota Cancer de l&#39;ovaire Alliance (MOCA) à MB. Les bailleurs de fonds ne jouaient aucun rôle dans la conception de l&#39;étude, la collecte et l&#39;analyse des données, la décision de publier ou de la préparation du manuscrit.

1. Préparation Lysis Buffer <ol><li> Dissoudre saccharose dans déminéralisée (DI) de l&#39;eau pour faire un bouillon solution 2 M. Filtrer une solution 2 M de saccharose en utilisant un vide poussé de 0,22 um polyfluorure de vinylidène (PVDF) filtre. </li><li> Dissoudre dithiothréitol (DTT) dans de l&#39;eau DI pour faire un mM solution et magasin 500 dans des conditions anaérobies. Dissoudre le chlorure de magnésium (MgCl 2) dans de l&#39;eau DI pour faire un bouillon solution 100 mM. Dissoudre l&#39;adénosine triphosphate (ATP) disodique hydraté dans de l&#39;eau DI pour faire un bouillon solution à 50 mM. </li><li> Préparer une solution de Tris à un pH de 7,4 en dissolvant le chlorhydrate de Trizma dans de l&#39;eau DI et à ajuster le pH avec de l&#39;hydroxyde de sodium (NaOH). </li><li> Moissonneuse parties des solutions mères dans de l&#39;eau désionisée pour faire un tampon de lyse avec une concentration en saccharose de 250 mM, une concentration de DTT de 1 mM, la concentration de MgCl2 5 mM, une concentration en ATP de 1 mM et une concentration de Tris de 50 mM . Par exemple, mélanger 535,3 ul de tris, 500 ul de MgCl2, 1,25 ml de saccharose, 20 ul de DTT, 200 ul d&#39;ATP et de 7,4947 ml d&#39;eau désionisée pour faire 10 ml de tampon de lyse. Ceci peut être utilisé pendant environ 20 expériences. </li></ol> 2. la culture de cellules pour l&#39;expérience <ol><li> Assurez médias par aliquotage 30 ml de DMEM + 10% de sérum fœtal bovin (FBS) dans un tube conique de 50 ml. </li><li> Transfert cellules HeLa M17 ou congelés de stockage d&#39;azote liquide à un petit bécher avec de l&#39;eau tiède. </li><li> Transférer les cellules à 50 ml le tube conique. D&#39;abord, ajouter certains médias à la fiole de cellule. Puis transférer les cellules remises en suspension dans les 5 secondes de la décongélation. </li><li> Centrifuger la suspension de cellules à 450 xg pendant 5 minutes pour obtenir un culot cellulaire. Ajouter 20 ml de milieu dans un flacon T75. </li><li> Retirez le support des cellules en utilisant aspiration. Ajouter 5 ml de milieu frais au culot cellulaire et remettre en suspension. </li><li> Transférer la suspension cellulaire 5 ml à 20 ml de milieu dans le flacon T75 et incuber à 37 ° C. ChAnge les médias tous les 3 jours. </li></ol> 3. Cellule récolte <ol><li> Retirez les médias par l&#39;intermédiaire aspiration, en faisant attention à ne pas toucher les cellules. </li><li> Laver les cellules avec 5 ml de solution saline tamponnée au phosphate (PBS). Retirer du PBS en utilisant une aspiration. </li><li> Ajouter 3 ml de trypsine-acide éthylènediaminetétra-acétique (EDTA) au flacon T75 et incuber à 37 ° C pendant 3 min. </li><li> Ajouter 6 ml de milieu frais dans le ballon et remettre en suspension les cellules. </li><li> Transférer la suspension dans un tube conique de 15 ml et centrifuger à 290 g pendant 5 minutes. </li><li> Eliminer le surnageant et ajouter 5 ml de PBS. Tapotez doucement le bas du tube pour briser le culot. Centrifuger à 290 g pendant 5 min. </li><li> Répétez l&#39;étape 3.6 trois fois. </li><li> Retirer du PBS et remettre les cellules dans 1 ml de PBS frais. Transfert au tube Eppendorf (1,5 ml). Spin à 290 g pendant 5 min. </li></ol> 4. lyse cellulaire <ol><li> Mesurer le volume approximatif de la pastille en utilisant une pipette d&#39;unD peser perles de verre dans une masse: rapport en volume de 1: 1. Ajouter deux fois le volume du culot dans un tampon de lyse. Note: Ajouter le tampon avant d&#39;ajouter les perles de verre. </li><li> Lyse des cellules dans le tube Eppendorf dans la agitation maximale pendant 30 minutes à 4 ° C par agitation par tourbillonnement dans une chambre froide. </li><li> Centrifuger à 200 g pendant 30 secondes pour régler les perles. Recueillir le surnageant. </li><li> Ajouter le même volume de billes de verre comme précédemment pour le surnageant. </li><li> Vortex nouveau à une agitation maximale pendant 30 min à 4 ° C. </li><li> Centrifuger brièvement à 200 xg pendant 30 sec à régler les perles de verre. Recueillir le surnageant. </li><li> Centrifuger à 5030 g pendant 5 min pour éliminer les noyaux, les membranes et les cellules non rompues. Récupérer le surnageant, ce qui est le lysat cellulaire. </li></ol> 5. L&#39;homogénéisation tissulaire <ol><li> Faites une masse: volume de 1: 9 solution de tissus de la moelle épinière de souris pour tampon de lyse. Remarque: Cette méthode est applicable à une variété de différerdes échantillons de tissus ENT. </li><li> Homogénéiser le tissu en utilisant un réglage de niveau 2 de l&#39;homogénéisateur pendant 30 s afin d&#39;assurer qu&#39;il n&#39;y a pas de morceaux restants. </li><li> Isoler à 5030 g pendant 3 min pour enlever les noyaux, les membranes et les cellules non rompues. </li><li> Récupérer le surnageant, qui est le lysat. </li></ol> 6. La dérivation de l&#39;échantillon <ol><li> Effectuer un acide bicinchoninique (BCA) dans une analyse de plaque de 96 puits pour déterminer la concentration en protéines du lysat de tissu en utilisant un lecteur de plaques à 96 puits colorimétrique comme spécifié par le Pierce BCA Protein Assay protocole de la trousse. </li><li> Amener une partie aliquote correspondant à 20 pg de protéine totale à l&#39;aide de 50 ul (DI) de l&#39;eau de-ionisée. </li><li> Ajouter 2 ul de 1,35 M de HA-Ub-vinyle sulfone (VS) à la solution et incuber pendant 1 heure à 37 ° C. Il en résulte 20 pg de lysat de 50 nM de sonde dans le mélange réactionnel final. Ceci est en grand excès molaire d&#39;assurer le marquage de DUBs fonctionnels. </li><li> JEncubate l&#39;échantillon dans le tampon d&#39;échantillon de Laemmli pendant 5 minutes à 95 ° C. Pour un volume de 52 ul de réaction utilisation 26 ul de tampon d&#39;échantillon 2x, 13 ul de tampon d&#39;échantillon 4x ou 8,87 pi de tampon d&#39;échantillon 6x. </li><li> Refroidir sur de la glace avant de charger le gel. </li></ol> 7. Western Blot <ol><li> Charger un 4-20% gel de tris-glycine 12 puits avec 40 pl de l&#39;échantillon préparé </li><li> Exécutez le gel à 95 V jusqu&#39;à ce que le front d&#39;ions atteint le fond. </li><li> Effectuer un transfert durant la nuit du gel sur un polyfluorure de vinylidène (PVDF). </li><li> Incuber la membrane dans la amido tache noire et de numériser l&#39;membane pour obtenir une image montrant la quantité de protéine dans chaque voie. </li><li> Destain la membrane et incuber dans 5% de lait dans du PBS pendant 1 heure pour bloquer. </li><li> Incuber la membrane dans l&#39;anticorps primaire anti-HA à une concentration de 1: 10000 dans 5% de lait dans du PBS, soit pendant une nuit à 4 ° C ou pendant 4 heures à température ambiante. </li><li> Effectuez 3 lavages fou 5 minutes chacun dans du PBS avec 0,1% de Tween-20 détergent. </li><li> Incuber la membrane à une peroxydase de raifort de la souris à une concentration de 1: 10000 dans 5% de lait dans du PBS pendant au moins 2 heures. </li><li> Effectuer trois lavages pendant 5 minutes chacun dans du PBS avec 0,1% de détergent Tween-20. </li><li> Effectuer 1 lavage pendant 5 min dans du PBS. </li><li> Incuber la membrane dans un réactif de détection de chimioluminescence pendant 10 min et la détection en utilisant un détecteur à chimiluminescence. Utilisez le réglage sur le détecteur automatique de l&#39;exposition. </li></ol>

<ol>
	<li>Ovaa, H., Kessler, B. M., Rol&#233;n, U., Galardy, P. J., Ploegh, H. L., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-+based+ubiquitin-specific+(USP)+profiling+of+virus-infected+and+malignant+human+cells.">Activity- based ubiquitin-specific (USP) profiling of virus-infected and malignant human cells.</a> Proc Natl Acad Sci USA. 101 (8), 2253-2258 (2004).</li><li>Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+deubiquitination:+Targeting+of+proteins+for+degradation+by+the+proteasome.">Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome.</a> Semin Cell Dev Biol. 11 (3), 141-148 (2000).</li><li>Ziad, M. E., Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+proteolysis+by+human+deubiquitinating+enzymes.">Regulation of proteolysis by human deubiquitinating enzymes.</a> Biochim Biophys Acta. 1843 (1), 114-128 (2014).</li><li>Rol&#233;n, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+Profiling+of+Deubiquitinating+Enzymes+in+Cervical+Carcinoma+Biopsies+and+Cell+Lines.">Activity Profiling of Deubiquitinating Enzymes in Cervical Carcinoma Biopsies and Cell Lines.</a> Mol Carcinog. 45 (4), 260-269 (2006).</li><li>Borodovsky, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry-Based+Functional+Proteomics+Reveals+Novel+Members+of+the+Deubiquitinating+Enzyme+Family.">Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family.</a> Chem Biol. 9 (10), 1149-1159 (2002).</li><li>Andersson, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+Parkinson&#8217;s-disease-associated+mutations+on+the+deubiquitinating+enzyme+UCH-L1.">The effect of Parkinson&#8217;s-disease-associated mutations on the deubiquitinating enzyme UCH-L1.</a> J Mol Biol. 407 (2), 261-272 (2011).</li><li>Poon, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Amyloid+(A&#946;)+oligomers+impair+brain-derived+neurotrophic+factor+retrograde+trafficking+by+down-regulating+ubiquitin+C-terminal+hydrolase+UCH-L1.">&#946;-Amyloid (A&#946;) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase UCH-L1.</a> J Biol Chem. 288 (23), 16937-16948 (2013).</li><li>Nijman, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP1+regulates+the+Fanconi+anemia+pathway.">The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.</a> Mol Cell. 17 (3), 331-339 (2005).</li><li>Stevenson, L. F., Sparks, A., Allende-Vega, N., Xirodimas, D. P., Lane, D. P., Saville, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP2a+regulates+the+p53+pathway+by+targeting+Mdm2.">The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.</a> EMBO J. 26 (4), 976-986 (2007).</li><li>Coughlin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+RA-9+inhibits+proteasome-associated+DUBs+and+ovarian+cancer+in+vitro+and+in+vivo+via+exacerbating+unfolded+protein+responses.">Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.</a> Clin Cancer Res. 20 (12), 3174-3186 (2014).</li><li>Colla, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+&#945;-Synucleinopathy+In.">Endoplasmic Reticulum Stress Is Important for the Manifestations of &#945;-Synucleinopathy In.</a> Vivo. J Neurosci. 32 (10), 3306-3320 (2012).</li></ol>

Les auteurs ont rien à révéler.

Depuis ubiquitination est une activité cellulaire fondamental, la compréhension des mécanismes de régulation pourrait être la clé pour déterrer les processus de la maladie et de la pathologie. L&#39;utilisation de HA marqué sites dirigé sondes actives Ub dérivés rapportés ici fournit une méthode simple, mais très applicable pour l&#39;étude de la dégradation des protéines Ub-médiation. Cette méthode est plus rapide et moins coûteux que d&#39;étudier chacun des DUBs individuellement. <p class="jo...

Le système ubiquitine-protéasome (UPS) sert comme l&#39;une des principales voies de dégradation dans la cellule de mammifère. Substrats liés à la dégradation dans le protéasome sont étiquetés de manière covalente avec des polymères de l&#39;ubiquitine (Ub) 1. Avant le substrat ciblé entre le protéasome de dégradation, l&#39;étiquette de poly-ubiquitine doit être retiré. Une classe d&#39;enzymes appelées enzymes de dé-ubiquitination (DUB) est responsable de l&#39;élimination et le recyclage des molécules d&#39;ubiquitine 2. Il a été prédite à partir du génome humain qui, il ya près d&#39;une centaine DUBs travaillant dans la cellule 3. Avec un si grand nombre de processus cellulaires DUBs contrôle Ub-médiation, l&#39;étude de ces enzymes représente un défi, car les techniques d&#39;ARNm ne donnent pas d&#39;informations sur l&#39;activité et western blot ne donne des informations sur les niveaux d&#39;expression. L&#39;utilisation de la grippe hémagglutinine (HA) marqué, Ub sondes dérivées de sites actifs dirigée permet un mod covalenteification des DUBs fonctionnels et donc donne une visualisation directe de l&#39;activité de ces enzymes sur un western blot 4. Les sondes ont un groupe réactif thiol C-terminal qui sert de substrat suicide pour le résidu cysteine ​​du site actif 5. Avec ces sondes, il est possible d&#39;étudier l&#39;activité et l&#39;expression de nombreux DUBs potentiel sous les deux états pathologiques et physiologiques de la cellule. Changements dans l&#39;activité DUB ont été impliqués dans une gamme de conditions pathologiques comme la maladie de Parkinson, la maladie d&#39;Alzheimer, l&#39;anémie et divers cancers 6-10. Cette technique fournit un outil puissant pour l&#39;étude des maladies. Dans le présent article, nous montrons l&#39;application de cette technique dans les cellules HeLa et M17 qui ont été lysées en utilisant des billes de verre. En outre, nous décrivons comment utiliser cette méthode dans des échantillons de tissus de la moelle épinière de souris. L&#39;information obtenue à partir de cette technique peut être utilisée comme un point de départ pour identifiercibles thérapeutiques ainsi que l&#39;établissement de modèles pour l&#39;étude des différents états pathologiques. La véritable utilité de cette technique réside dans sa capacité à fournir des informations sur plusieurs DUBs dans un seul essai.

<table border="0" cellpadding="0" cellspacing="0" style="width:615px;" width="615">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PowerGen 125 ( Homogenizer)</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">14-261-02P</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum-driven filters .22&#181;M&#160;</td>
			<td style="width:113px;"></td>
			<td style="width:119px;">BPV2210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed &#8804;106&#181;m (~140 U.S. sieve)</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">G4649-10G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">S6-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adenosine 5&#39;-triphosphate disodium salt hydrate</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">A26209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma hydrochloride&#160;</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">T3253</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture flask 75cm2 w/ filter cup 250 ml 120/ cs</td>
			<td style="width:113px;">Cellstar</td>
			<td style="width:119px;">T-3001-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HI FBS</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">16140-071</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-HA antibody produced in mouse</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">H9658</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ubiquitin vinyl sulfone (HA-tag)</td>
			<td style="width:113px;">Enzo Life Sciences</td>
			<td style="width:119px;">BML-UW0155-0025</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laemmli&#39;s SDS-Sample Buffer (4X, reducing)</td>
			<td style="width:113px;">Boston BioProducts</td>
			<td style="width:119px;">BP-110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:113px;">ThermoScientific</td>
			<td align="right" style="width:119px;">23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples

Le système ubiquitine-protéasome a été récemment impliqué dans différentes pathologies dont les maladies neurodégénératives et des cancers. À la lumière de cela, des techniques pour étudier le mécanisme de régulation de ce système sont essentielles pour élucider les processus cellulaires et moléculaires des maladies susmentionnées. L&#39;utilisation de l&#39;hémagglutinine dérivée d&#39;ubiquitine sondes décrites dans le présent document constitue un outil précieux pour l&#39;étude de ce système. Ce document décrit en détail une méthode qui permet à l&#39;utilisateur d&#39;effectuer des tests qui donnent une visualisation directe de l&#39;activité enzymatique désubiquitination. Enzymes de dé-ubiquitination contrôlent dégradation par le protéasome et partager homologie fonctionnelle sur leurs sites actifs, ce qui permet à l&#39;utilisateur d&#39;enquêter sur l&#39;activité de plusieurs enzymes dans un essai. Lysats sont obtenus grâce à une rupture mécanique doux cellulaire et incubées avec site actif sondes dirigées. Enzymes fonctionnelles sont étiquetés avec les sondes en enzymes inactives demeurent non. En termeNing cet essai, l&#39;utilisateur obtient des informations à la fois sur l&#39;activité et l&#39;expression potentielle de plusieurs enzymes de dé-ubiquitination d&#39;une manière rapide et facile. La méthode actuelle est nettement plus efficace que d&#39;utiliser des anticorps individuels pour les cent prédits enzymes de dé-ubiquitination dans la cellule humaine.

Cellules M17 et HeLa cultivées ont été récoltées en utilisant la méthode détaillée dans le (Cell récolte 3.) protocole et les tissus de la moelle épinière de souris a été obtenu. L&#39;/ tissu de la moelle épinière culot cellulaire a été placé dans un tube avec le tampon de lyse décrit dans la section de préparation des réactifs. Les culots cellulaires ont été lysés en utilisant des billes de verre (Figure 1A) et des échantillons de tissus de moelle épinière de souris ont ét?...

Watch this Scientific Journal Video about Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples at JoVE.com

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Le protocole actuel détaille un procédé pour mesurer l&#39;activité des enzymes fonctionnellement homologue désubiquitination. Sondes spécialisées modifier de manière covalente l&#39;enzyme et permettent de détecter. Cette méthode a le potentiel d&#39;identifier de nouvelles cibles thérapeutiques.

Méthode de mesure de l&#39;activité des enzymes de dé-ubiquitination dans des lignées cellulaires et des échantillons de tissus

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

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9.5K vues.  University of Minnesota.  Le protocole actuel détaille un procédé pour mesurer l'activité des enzymes fonctionnellement homologue désubiquitination. Sondes spécialisées modifier de manière covalente l'enzyme et permettent de détecter. Cette méthode a le potentiel d'identifier de nouvelles cibles thérapeutiques. 

Vidéo: Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Co-actine sédimentation Assay; pour l&#39;analyse des Binding Protein à F-actine

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

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Biologie

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

La quantification de γH2AX Foci dans les échantillons de tissus

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Un test pour mesurer l&#39;activité des Escherichia coli Inductible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

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 dispositif de récupération d&#39;échantillons cryoconservés tombé accidentellement dans les réservoirs de stockage cryogéniques

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 ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Mesure<em&gt; In vitro</em&gt; Activité ATPase pour Enzymatic Caractérisation

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Comparaison de trois différentes méthodes pour la détermination de la prolifération cellulaire dans des lignées cellulaires de cancer du sein

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Mesure de l’activité enzymatique Lactase dans le laboratoire d’enseignement

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Une méthode Sectionnement améliorée pour les échantillons de tissus végétaux récalcitrantes

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

Dissection de l&#39;Auditory Bulla chez les souris postnatale: Isolation des os de l&#39;oreille moyenne et l&#39;analyse histologique

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear : Un tissu méthode de compensation pour l’imagerie en trois dimensions du tissu adipeux

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...