Nous remercions Simon Alberti pour l&#39;assistance à l&#39;élaboration de ce protocole. Ce travail a été soutenu par une subvention des National Institutes of Health (GM25874), du Howard Hughes Medical Institute Investigatorship (pour SL), et un National Science Foundation bourse de formation pré-doctorale (pour HR).

<html> Partie 1: Préparer le gel <ol><li> Montez le plateau de coulée de gel. L&#39;équipement standard pour l&#39;électrophorèse l&#39;ADN horizontale peut être utilisé. Pour un grand nombre d&#39;échantillons, nous préparons un 20 cm x 24 cm dalle avec un maximum de quatre 50-bien peignes. Assurez-vous que la dalle n&#39;a pas les égratignures, car ils peuvent déformer l&#39;image blot. </li><li> Créer une solution de 1,5% d&#39;agarose (moyen ou élevé de gel solidité, faible OEE) de TAE 1X. Le volume devrait être suffisant pour submerger entièrement les dents du peigne - vous voulez charger l&#39;échantillon autant que possible pour maximiser la détection. Micro-ondes le mélange jusqu&#39;à l&#39;agarose est complètement dissous. </li><li> Ajouter rapidement SDS à 0,1% à partir d&#39;un stock de 10%. Faire tourbillonner pour mélanger. Si certains d&#39;agarose se solidifie au cours de cette étape, redissolvent utilisant une plaque chauffante et être prudent pour éviter l&#39;ébullition. </li><li> Verser la solution dans le bac prépondérante. Utilisez un peigne à ratisser les bulles, car ils pourraient interférer avec le transfert tard. </li><li> Après un gel a pris, retirer peignes et placer le gel dans le réservoir de gel. Immerger complètement le gel dans 1X TAE contenant 0,1% SDS. </li></ol> Partie 2: Préparation des échantillons <ol><li> Pour analyse à haut débit des lysats levure, nous commençons avec 2 ml cultures cultivées pendant une nuit sous agitation rapide de 96 puits blocs. Dans ce cas, chaque culture est surexprimant une protéine d&#39;intérêt. Lors de l&#39;analyse des protéines de faible abondance, les volumes de culture plus large doit être utilisé </li><li> Récolte des cellules par centrifugation à 2000 RCF pendant 5 min à température ambiante. </li><li> Resuspendre les cellules dans l&#39;eau et centrifuger à nouveau. </li><li> Remettre en suspension dans 1 ml de solution sphéroplastes. Incuber pendant environ 30 min à 30 ° C (vous pouvez confirmer l&#39;efficacité sphéroplastes par microscopie). </li><li> Centrifuger à 800 RCF pendant 5 min à température ambiante. Supprimer complètement le surnageant. </li><li> Resuspendre sphéroplastes granulés dans 100 ml de tampon de lyse. </li><li> Couvrir le bloc avec du ruban adhésif et le vortex à haute vitesse pendant 2 min. </li><li> Culot cellulaire débris à 4000 RCF pendant 2 min. </li><li> Retirer délicatement le surnageant dans un nouveau conteneur, par exemple, une plaque de 96 puits PCR. </li><li> Si désiré, déterminer la concentration en protéines des lysats. </li><li> Ajouter le tampon d&#39;échantillon 4X pour les échantillons contenant de générer lysats 1X tampon de l&#39;échantillon. Incuber pendant 5 min à température ambiante. </li><li> Charge de gel. Si désiré, économiser la moitié du volume d&#39;échantillon et de la faire bouillir pour analyse SDS-PAGE. Afin de surveiller l&#39;ampleur du transfert par la suite, notamment un pré-SDS-PAGE coloré marqueur. De plus, un marqueur de poids moléculaire composé de protéines très élevé (par exemple, le poulet pectoral extrait) peut être utilisé pour estimer la taille des complexes résolus. </li><li> Exécutez à basse tension (≤ 3 la longueur du gel V / cm) jusqu&#39;à ce que le front atteigne ~ 1 cm de l&#39;extrémité du gel. Cela va prendre plusieurs heures. Il est important que le gel rester cool, sinon la diffusion de protéines de faible poids moléculaire (par exemple, les monomères) peut limiter leur détection. </li></ol> Partie 3: Transfert <ol><li> Coupez un morceau de nitrocellulose aux mêmes dimensions que le gel. </li><li> Coupez 20 morceaux de GB004 et GB002 8 morceaux de papier buvard, pour les mêmes dimensions que le gel. Couper une pièce supplémentaire de GB004 pour être utilisé comme une mèche, il font environ 20 centimètres plus large que le gel. </li><li> Plonger la nitrocellulose, mèche, et 4 morceaux de GB002 en 1X SCT. </li><li> Dans un récipient en plastique, d&#39;assembler une pile de papiers comme suit: 20 morceaux de GB004 sécher, puis 4 morceaux de GB002 sec, puis un morceau de pré-humide GB002. Poser la nitrocellulose en haut de cette pile. </li><li> Rincer le gel sur le plateau de coulée brièvement dans l&#39;eau pour enlever l&#39;excès tampon. Puis, soigneusement commencer à glisser le gel hors du bac sur la pile. Tout en faisant glisser le gel hors du bac, éteindre la membrane avec le SCT, si nécessaire. Le tampon supplémentaire permet d&#39;éviter les bulles de devenir coincé sous le gel. Une pipette de transfert fonctionne bien dans ce but. </li><li> Après le gel a été déplacé à la pile, vérifiez soigneusement pour les bulles. Si elles existent, soulevez le bord du gel et de réappliquer tampon jusqu&#39;à ce que les bulles peuvent être élaborées. </li><li> Mettez les trois autres pré-mouillé GB002 morceaux sur le dessus du gel. Assurer un contact complet entre toutes les couches de roulement par une pipette fermement sur le haut de la pile. </li><li> Flanc la pile de transfert avec deux plateaux élevés contenant du SCT. Drapé de la mèche pré-humide dans la pile de sorte que chaque extrémité de la mèche est immergé dans le SCT. </li><li> Couvrir la pile transfert assemblé avec un plateau en plastique supplémentaires portant le poids supplémentaire (par exemple, une bouteille de 500 ml d&#39;eau). </li><li> Autoriser le transfert de procéder à un minimum de trois heures, ou jusqu&#39;au lendemain. </li><li> Après le transfert, la membrane peut être traitée par la norme Western blot. </li></ol> Solution sphéroplastes 1,2 M de D-sorbitol 0,5 mM MgCl 2 Tris 20 mM, pH 7,5 50 mM BME (ajouter frais) 0,5 mg / ml Zymolyase 100T (ajouter frais) coration: underline; &quot;Lysis Buffer&gt; 100 mM de Tris 7,5 NaCl 50 mM BME 10 mM (ajouter frais) inhibiteurs de protéase (ajouter frais) Tampon d&#39;échantillon 4X 2X TAE 20% de glycérol 8% de SDS bleu de bromophénol à la préférence

<ol>
	<li>Kryndushkin, D. S., Alexandrov, I. M., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+[PSI+]+prion+aggregates+are+formed+by+small+Sup35+polymers+fragmented+by+Hsp104.">Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104.</a> J. Biol. Chem. 278, 49636-49643 (2003).</li><li>Alexandrov, I. M., Vishnevskaya, A. B., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+and+Propagation+of+Polyglutamine-based+Amyloids+in+Yeast:+TYROSINE+RESIDUES+ENABLE+POLYMER+FRAGMENTATION.">Appearance and Propagation of Polyglutamine-based Amyloids in Yeast: TYROSINE RESIDUES ENABLE POLYMER FRAGMENTATION.</a> J. Biol. Chem. 283, 15185-15192 (2008).</li><li>Allen, K. D., 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=Hsp70+chaperones+as+modulators+of+prion+life+cycle:+novel+effects+of+Ssa+and+Ssb+on+the+Saccharomyces+cerevisiae+prion+[PSI+].">Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].</a> Genetics. 169, 1227-1242 (2005).</li><li>Aron, R., Higurashi, T., Sahi, C., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J-protein+co-chaperone+Sis1+required+for+generation+of+[RNQ+]+seeds+necessary+for+prion+propagation..">J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation..</a> The EMBO journal. 26, 3794-3803 (2007).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+amyloid+aggregates+using+agarose+gel+electrophoresis.">Analysis of amyloid aggregates using agarose gel electrophoresis.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Borchsenius, A. S., Muller, S., Newnam, G. P., Inge-Vechtomov, S. G., Chernoff, Y. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+variant+maintained+only+at+high+levels+of+the+Hsp104+disaggregase.">Prion variant maintained only at high levels of the Hsp104 disaggregase.</a> Current Genetics. 49, 21-29 (2006).</li><li>Salnikova, A. B., Kryndushkin, D. S., Smirnov, V. N., Kushnirov, V. V., Ter-Avanesyan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsense+suppression+in+yeast+cells+overproducing+Sup35+(eRF3)+is+caused+by+its+non-heritable+amyloids.">Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids.</a> J. Biol. Chem. 280, 8808-8812 (2005).</li><li>Tank, E. M., Harris, D. A., Desai, A. A., True, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+protein+repeat+expansion+results+in+increased+aggregation+and+reveals+phenotypic+variability.">Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability.</a> Mol. Cell. Biol. 27, 5445-5455 (2007).</li><li>Douglas, P. 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=Chaperone-dependent+amyloid+assembly+protects+cells+from+prion+toxicity.">Chaperone-dependent amyloid assembly protects cells from prion toxicity.</a> Proc. Natl. Acad. Sci. USA. 105, 7206-7211 (2008).</li><li>Nagy, B., Costello, R., Csako, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downward+blotting+of+proteins+in+a+model+based+on+apolipoprotein(a)+phenotyping.">Downward blotting of proteins in a model based on apolipoprotein(a) phenotyping.</a> Analytical Biochemistry. 231, 40-45 (1995).</li></ol>

SDD-AGE a été d&#39;abord rapporté par Kryndushkin et al. 1, pour étudier SDS-résistants complexes de la [PSI +] prion chez la levure, et a depuis trouvé une utilisation largement répandue étudier à la fois à prions et les non-prion agrégats 2-9. Toutefois, le transfert des protéines à une électrophorèse membrane suite à un gel d&#39;agarose est problématique, et peut aboutir à une image déformée blot 5. De plus, la technique submergée électrotransfert les plus couramment utilisés introduit des limitations pratiques à la taille du gel et donc le nombre d&#39;échantillons qui peuvent être traitées. Nous avons résolu ces problèmes en employant la baisse transfert capillaire 10, une procédure simple qui utilise une pile de sèche buvards de transférer les protéines du gel sur une membrane de nitrocellulose. Transfert capillaire évite la déformation et permet des gels importants pour être traités facilement. Il ya quelques choses à considérer avant d&#39;utiliser DDS-AGE. Pour les échantillons bruts (par exemple, des lysats), immunodétection des protéines spécifiques est nécessaire. SDD-âge n&#39;a pas totalement dénaturer les complexes protéines d&#39;intérêt, alors la protéine (s) à détecter doit porter une étiquette épitopique en dehors de la région amyloïdogénique. Lysats peuvent généralement être préparés comme ils le seraient pour une normale de SDS-PAGE, avec deux différences importantes. Premièrement, les soins accrue doivent être prises pour prévenir la dégradation par protéolyse. Les conditions partiellement dénaturantes utilisées ici ne sont pas suffisantes pour inactiver les protéases, et peut également fabriquer des protéines cibles plus sensibles à la protéolyse. Utilisez un cocktail inhibiteur de la protéase complète au moins deux fois la concentration recommandée. Deuxièmement, le chauffage des échantillons devrait être évitée. Si un contrôle tous les monomères négatif est souhaitée, par exemple pour confirmer que haut poids moléculaire des espèces ne sont pas dues à des modifications covalentes, une incubation de 10 minutes à 95 ° C peut être utilisé, ce qui va restaurer la plupart amyloïdes à la protéine monomérique.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>zymolyase 100T</td>
<td>&nbsp;</td>
<td>Seikagaku America</td>
<td>120493-1</td>
<td>&nbsp;</td>
<tr>
<td>Halt Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>78429</td>
<td>&nbsp;</td>
<tr>
<td>Hybond-C extra nitrocellulose</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN303E</td>
<td>&nbsp;</td>
<tr>
<td>GB004 blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>10427926</td>
<td>&nbsp;</td>
<tr>
<td>GB002 (3MM Chr) blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-917</td>
<td>&nbsp;</td>
<tr>
<td>Agarose (UltraPure)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15510-027</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis

L&#39;agrégation amyloïde est associée à de nombreuses pathologies mauvais repliement des protéines et des sous-tend les propriétés infectieuses des prions, qui sont auto-conformation des protéines templating qui sont pensés pour avoir des rôles bénéfiques dans les organismes inférieurs. Amyloïdes ont été notoirement difficile à étudier en raison de leur insolubilité et de l&#39;hétérogénéité structurelle. Cependant, la résolution de polymères amyloïde basée sur la taille et de l&#39;insolubilité de détergent a été rendue possible par semi-dénaturantes Détergent-électrophorèse sur gel (SDD-âge). Cette technique est de trouver l&#39;utilisation généralisée pour la détection et la caractérisation des variantes amyloïdes conformation. Ici, nous démontrons une adaptation de cette technique qui facilite son utilisation dans les applications à grande échelle, tels que des écrans pour les prions roman et d&#39;autres protéines amyloïdogénique. La nouvelle SDD-ÂGE méthode utilise le transfert capillaire pour une plus grande fiabilité et facilité d&#39;utilisation, et permet à tout gel de taille à être logé. Ainsi, un grand nombre d&#39;échantillons, préparés à partir de cellules ou de protéines purifiées, peuvent être traitées simultanément la présence de SDS-insolubles de protéines conformères marqués.

Watch this Scientific Journal Video about Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis at JoVE.com

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

DDS-AGE est une technique utile pour la détection et la caractérisation de l&#39;amyloïde comme les polymères dans les cellules. Ici nous démontrons une adaptation qui rend cette technique prêtent à grande échelle des applications.

Le dépistage de l&#39;agrégation amyloïde par les semi-électrophorèse sur gel dénaturant Détergent-Agarose

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

screening-for-amyloid-aggregation-semi-denaturing-detergent-agarose

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26.7K vues.  Whitehead Institute for Biomedical Research. DDS-AGE est une technique utile pour la détection et la caractérisation de l'amyloïde comme les polymères dans les cellules. Ici nous démontrons une adaptation qui rend cette technique prêtent à grande échelle des applications.

Vidéo: Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Électrophorèse sur gel dénaturant Dégradé (DGGE)

Recherche

Biologie

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Électrophorèse sur gel dénaturant Urée polyacrylamide (PAGE urée)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

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

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

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

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

Bleu d&#39;électrophorèse sur gel de polyacrylamide natif (BN-PAGE) pour l&#39;analyse des complexes multiprotéiques de lysats cellulaires

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

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

Verser et exécution d&#39;un gel de protéine en réutilisant Cassettes commerciaux

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Électrophorèse sur gel d&#39;agarose pour la séparation de fragments d&#39;ADN

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Mucine Agarose Gel Electrophoresis: Western Blot pour les glycoprotéines de haut poids moléculaire,


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Méthodes d’étude des changements dans l’agrégation des protéines inhérente avec l’âge chez Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Agrégation de récepteur de Surface cellulaire microscopie confocale révèle par spectroscopie de corrélation d’Image

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Analyse des Complexes de chaîne respiratoire mitochondriale dans les cellules humaines cultivées à l’aide de bleu sur Native de Gel de Polyacrylamide et Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...