Wir danken Simon Alberti für die Unterstützung bei der Entwicklung dieses Protokolls. Diese Arbeit wurde durch ein Stipendium der National Institutes of Health (GM25874), ein Howard Hughes Medical Institute Investigatorship (zum SL), und ein National Science Foundation Chemiefonds Ausbildungsförderung (RH) unterstützt.

<html> Teil 1: Vorbereiten des Gels <ol><li> Montieren Sie den Gelträger. Serienausstattung für horizontale DNA-Elektrophorese verwendet werden kann. Für eine große Anzahl von Proben, bereiten wir eine 20 cm x 24 cm Platte mit bis zu vier 50-gut Kämme. Sicherstellen, dass die Platte nicht Kratzer, da diese die blot Bild verzerren können. </li><li> Erstellen Sie ein 1,5% Agarose-Lösung (mittlere oder hohe Gel-Festigkeit, geringes EBO) in 1X TAE. Das Volumen sollte ausreichen, um komplett versenken die Kammzähne - Sie wollen so viel wie möglich zu laden Probe den Nachweis zu maximieren. Microwave die Mischung, bis die Agarose vollständig gelöst ist. </li><li> Schnell add SDS zu 0,1% aus einer 10% Aktien. Swirl zu mischen. Wenn einige Agarose erstarrt bei diesem Schritt wieder auflösen mit einer heißen Platte und vorsichtig sein, um zu vermeiden Kochen. </li><li> Füllen Sie die Lösung in die Casting-Fach. Verwenden Sie einen Kamm zu harken jegliche Blasen, wie sie später mit Transfer stören könnten. </li><li> Nach gel gesetzt hat, entfernen Sie Kämme und Stelle das Gel in das Gel Tank. Ganz untertauchen das Gel in 1X TAE mit 0,1% SDS. </li></ol> Teil 2: Probenvorbereitung <ol><li> Für Hochdurchsatz-Analyse von Hefe-Lysaten, beginnen wir mit 2-ml-Kulturen über Nacht unter schnellem Rühren in 96-Well-Blocks gewachsen. In diesem Fall wird jede Kultur Überexpression eines Proteins von Interesse. Bei der Analyse geringer Menge Proteine, müssen größere Kultur-Volumes verwendet werden </li><li> Ernten Sie die Zellen durch Zentrifugation bei 2000 RCF für 5 min bei Raumtemperatur. </li><li> Die Zellen in Wasser und Zentrifuge wieder. </li><li> Resuspendieren in 1 ml Spheroplasting Lösung. Inkubieren für ca. 30 min bei 30 ° C (Sie können Spheroplasting Effizienz durch Mikroskopie bestätigt). </li><li> Zentrifuge bei 800 RCF für 5 min bei Raumtemperatur. Vollständig zu entfernen Überstand. </li><li> Resuspendieren pelletiert Sphäroplasten in 100 ml Lysepuffer. </li><li> Bedecken Sie den Block mit Klebeband und Wirbel bei hoher Geschwindigkeit für 2 min. </li><li> Pellet Zelltrümmer bei 4000 RCF für 2 min. </li><li> Sorgfältig Überstand zu entfernen, um einen neuen Container, z. B. eine 96-well PCR-Platte. </li><li> Wenn gewünscht, Bestimmung der Proteinkonzentration der Lysate. </li><li> Add 4X Probenpuffer, um die Proben zu Lysaten mit 1x Probenpuffer zu generieren. Inkubieren für 5 min bei Raumtemperatur. </li><li> Last-Gel. Falls gewünscht, speichern Sie die Hälfte des Probenvolumens und kochen sie für die SDS-PAGE-Analyse. Um das Ausmaß der Übertragung später auf dem Monitor, eine pre-gefärbten SDS-PAGE-Marker. Darüber hinaus kann ein Molekulargewichtsmarker, bestehend aus sehr großen Proteine ​​(zB Huhn pectoralis-Extrakt) verwendet, um Größen von der beschlossenen Komplexe zu schätzen. </li><li> Run bei niedriger Spannung (≤ 3 V / cm Gel Länge) bis der Farbstoff vor Erreichen ~ 1 cm vom Ende des Gels. Dies wird mehrere Stunden dauern. Es ist wichtig, dass das Gel kühl bleiben, sonst Diffusion von niedermolekularen Proteins (z. B. Monomere) können ihre Nachweisgrenze. </li></ol> Teil 3: Transfer <ol><li> Schneiden Sie ein Stück Nitrozellulose, um die gleichen Abmessungen wie das Gel. </li><li> Cut 20 Stück GB004 und GB002 8 Stück Löschpapier, auf die gleichen Abmessungen wie das Gel. Cut ein zusätzliches Stück GB004 als Docht verwendet werden; es etwa 20 Zentimeter breiter als das Gel. </li><li> Tauchen Sie die Nitrozellulose, Docht und 4 Stück GB002 in 1X TBS. </li><li> In einem Kunststoff-Behälter, montieren Sie einen Stapel von Papieren wie folgt: 20 Stück trockenen GB004, dann 4 Stück trockenes GB002, dann ein Stück vor nassen GB002. Legen Sie die Nitrozellulose auf dieses Stacks. </li><li> Spülen Sie das Gel auf die Gelträger kurz in Wasser, um überschüssiges Laufpuffer zu entfernen. Dann vorsichtig beginnen, um das Gel vor der Tablettrutsche auf den Stapel. Während Sie das Gel aus dem Fach, begießen die Membran mit TBS wie nötig. Die zusätzlichen Puffer verhindert, dass Luftblasen aus Einklemmen unter dem Gel. Ein Transferpipette eignet sich gut für diesen Zweck. </li><li> Nachdem das Gel wurde auf den Stapel verschoben worden ist, sorgfältig überprüfen, ob Luftblasen. Falls vorhanden sind, heben Sie den Rand des Gels und erneut Puffer, bis die Blasen ausgearbeitet werden kann. </li><li> Legen Sie die restlichen drei vorbefeuchtet GB002 Stücke auf dem Gel. Auf gute Kontakt zwischen allen Schichten durch Walzen einer Pipette fest an der Oberseite des Stapels. </li><li> Flank die Übertragung Stack mit zwei erhöhten die Fächer mit TBS. Hängen Sie die Pre-wet Docht über den Stack, so dass jedem Ende der Docht in TBS getaucht ist. </li><li> Decken Sie die versammelten Transfer-Stack mit einem zusätzlichen Kunststoff-Tablett, auf dem Zusatzgewicht (zB eine 500 ml Flasche Wasser). </li><li> Lassen Sie den Transfer für ein Minimum von drei Stunden oder über Nacht gehen. </li><li> Nach der Übertragung kann die Membran durch Standard-Western-Blot-verarbeitet werden. </li></ol> Spheroplasting Lösung 1,2 M D-Sorbit 0,5 mM MgCl 2 20 mM Tris, pH 7,5 50 mM BME (add frisch) 0,5 mg / ml Zymolyase 100T (add frisch) coration: underline; &quot;&gt; Lysispuffer 100 mM Tris 7,5 50 mM NaCl 10 mM BME (add frisch) Protease-Inhibitoren (add frisch) 4X Sample Buffer 2X TAE 20% Glycerin 8% SDS Bromphenolblau den Vorzug

<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 wurde zuerst von Kryndushkin et al. 1, zu SDS-resistenten Komplexen des Studiums der [PSI +] Prion in Hefe, und hat seitdem große Verbreitung gefunden Studium sowohl Prion-und Nicht-Prion-Aggregate 2-9. Allerdings ist die Übertragung der Proteine ​​auf eine Membran nach Elektrophorese in einem Agarosegel problematisch und kann zu einem verzerrten Bild 5 blot Ergebnis. Darüber hinaus führt die versunkene Elektroblotting Technik am häufigsten verwendeten praktischen Grenzen für die Größe des Gels und damit die Anzahl der Proben, die verarbeitet werden können. Wir haben diese Probleme durch den Einsatz von nach unten Kapillartransfer 10, ein einfaches Verfahren, das einen Stapel von Dry-Blotting Papiere benutzt, um Proteine ​​aus dem Gel auf eine Nitrozellulosemembran übertragen gerichtet. Kapillar-Transfer verhindert Verzerrungen und ermöglicht große Gele auf einfache Weise bearbeitet werden. Es gibt ein paar Dinge, die vor über SDD-AGE zu betrachten. Für Rohöl Proben (zB Lysate), wird durch immunologischen Nachweis spezifischer Proteine ​​notwendig. SDD-AGE nicht vollständig denaturiert das Protein-Komplexe von Interesse, so das Protein (s) nachgewiesen werden muss ein Epitop-Tag außerhalb der amyloidogene Region zu tragen. Lysate können in der Regel hergestellt, wie sie für eine normale SDS-PAGE werden würde, mit zwei wichtigen Unterschieden. Zunächst muss darauf geachtet werden, erhöht den Abbau durch Proteolyse zu verhindern. Die teilweise denaturierenden Bedingungen werden dabei nicht ausreichend, um Proteasen zu inaktivieren, und kann auch Zielproteine ​​anfälliger für Proteolyse. Verwenden Sie eine komplette Protease-Inhibitor-Cocktail an mindestens zwei-fache der empfohlenen Konzentration. Zweitens Erwärmung der Proben sollte vermieden werden. Wenn ein all-Monomer negative Kontrolle ist erwünscht, um beispielsweise sicherzustellen, dass hohe Molekulargewicht Arten nicht durch kovalente Modifikationen, eine 10-minütige Inkubation bei 95 ° C eingesetzt werden können, die die meisten Amyloide auf monomeres Protein wird wieder herzustellen.

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

Amyloid-Aggregation ist mit zahlreichen Proteinfehlfaltung Pathologien und unterliegt der infektiösen Eigenschaften von Prionen, die konformativ sind selbsterklärend Templating Proteine, die vermutlich von Vorteil Rollen in niederen Organismen haben. Assoziiert Amyloide sind notorisch schwierig zu studieren aufgrund ihrer Unlöslichkeit und strukturelle Heterogenität. Allerdings hat eine Auflösung von Amyloid-Polymere auf Basis von Größe und Reinigungsmittel Unlöslichkeit Basis wurde möglich durch Semi-denaturierenden Detergens-Agarose-Gel-Elektrophorese (SDD-AGE). Diese Technik ist zu finden breite Anwendung für den Nachweis und die Charakterisierung von Amyloid-Konformations-Varianten. Hier zeigen wir, eine Anpassung dieser Technik, dass seine Verwendung erleichtert in großen Anwendungen, wie zum Beispiel Bildschirme für neuartige Prionen und anderen amyloidogenen Proteinen. Das neue SEPA-AGE-Methode verwendet Kapillartransfer für höchste Zuverlässigkeit und Benutzerfreundlichkeit und ermöglicht Unternehmen jeder Größe Gel untergebracht werden. So kann eine große Anzahl von Proben, die aus Zellen oder gereinigte Proteine ​​hergestellt, gleichzeitig auf das Vorhandensein von SDS-unlöslichen Konformere markierte Proteine ​​weiterverarbeitet werden.

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

SDD-AGE ist eine nützliche Technik für die Erkennung und Charakterisierung von Amyloid-ähnlichen Polymeren in den Zellen. Hier zeigen wir eine Anpassung, dass diese Technik zugänglich großflächige Anwendungen macht.

Screening für Amyloid-Aggregation durch Semi-denaturierenden Detergens-Agarose-Gel-Elektrophorese

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

Nanyang Technological University

Research

JoVE Journal

Biology

26.9K Aufrufe.  Whitehead Institute for Biomedical Research. SDD-AGE ist eine nützliche Technik für die Erkennung und Charakterisierung von Amyloid-ähnlichen Polymeren in den Zellen. Hier zeigen wir eine Anpassung, dass diese Technik zugänglich großflächige Anwendungen macht.

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

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio film communities. So I am profiling the bacterial communities by looking at their 60 N sequence and how this sequence changes over time in space. And to do that, I'm using DDGE.This is not a normal acry of my well, it needs to be kept at 60 degrees when it's run and the buffer needs to be pumped continuously. So we're gonna warm up the buffer for about an hour until it's at 60 degrees, and then we can run the towel. Now I'm gonna wash this plate with dilute uc solution, peeling around for impurities, andone, light that up.Anything like that can cause regularities. It is important to wipe off all the water. So when I assemble the gel, I'll put that chip on the outside of the gel.So the gel will be in between these two pieces of glass and the chip will be on the outside and it won't affect the gel in between. So now I'm gonna assemble the gel in these clamps and then I squeeze them together and tighten by turning to the right. As with any gel, it's important to make sure that the bottom is flush.To prevent making, we now turn the gel around and put it in here to get sealed with the gasket. So it's important when you do this that the gel, that the plates are actually lined up with the gasket here. So these knobs on the side then go in and you press down to type the seal.So this is a 30 mil syringe. And this part and this part come with the bio red kit and it just on like this. And that's just to get rid of the excess water in the tubes.So put the tubing onto the gel to load the gel. First you put an adapter into the tubing and then a needle look with that small adapter and put it right in the center of the gel just so it's fairly below the bottom glass. Then this wire adapter goes into the long tubing and it's going to connect to equal syringes containing the high and the low density solution.So these are pre-prepared solutions. The blue one, I've added a dye here that just indicates that this is the most denaturing solution and this is the least fitting train solution, the low density one. And they have everything in them.And now I'm gonna to make, gonna be Actually I twice as much the week. And once you've got the tempt, you have about five to seven minutes to fill the gel before the colon rises. And now we fill the syringe juice with the solution.So I'm gonna fill it with about 15 the solution. And then to get rid of any air bubbles, I'll turn it upside down and squeeze gently until that top air bubble is gone. Now this little screw here is gonna go into this groove.This adapter is gonna line up with this clamp here, and it's gonna get C clamped in very tightly. And then this tubing is gonna attach to the syringe. And the same thing is done on the other side with the high density matrix where it's time to deliver.And so I'm gonna be turning this wheel in a steady clockwise motion. And first the blue solution is gonna go out first because that's the the most in nature, and it's gonna form the bottom part of the gel. And then after a little while, the clear solution will start filling in the gel and the Gradient will be formed.One moment is the gently on an angle to And as soon as you finish pouring the gel to wash out your tubes immediately because they will polymerize Going, It's the group there. And then press down. So this little, And on the other side, I'll do the same thing.Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio communities. So I am profiling the bacterial communities by looking at their 60 F sequence and how the sequence changes over time in space. And to do that, I'm using DDGE.So DGE is Dena gel electrophoresis. And basically the idea is that there's a gradient and the DNA, as it runs down the gradient gets de natured. And so different 16 species will stop at different points along the gradient.And so it ends up being a fingerprint of the different sexiness species in the community. So the goal of my experiment is to see who is in the community and how that's changing over time and space. And I can run the sexiness DDD gel and then I can cut out the bands and identify the species that are represented in each community.So one of the biggest issues when, when doing A DDG gel is to figure out the gradient that's the best. And so that is just something that needs to be played around with and figuring out for a particular community which type of gradient. So the one I'm using today is 35 to 55%urea.One of the big problems that can happen with this technique is that smears come up in in the leans. And I found for that, for whatever reason, one of the best things to do is to run the gels for a really long time and then the smears disappear. Well, today I'm just using it for general bacterial communities, but this can also be used for a specific family of bacteria to see how those change over time, and those tend to be very clean gels.You can also look at mutations in single genes with DDGE.

Denaturierende Gradienten-Gelelektrophorese (DGGE)

Forschung

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.

Denaturierung Urea Polyacrylamidgelelektrophorese (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 ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Live-Präparation Drosophila Embryos: Optimierte Verfahren für das Screening Mutant Sammlungen von Antikörper-Färbung

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

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.

Blaue Native Polyacrylamid-Gelelektrophorese (BN-PAGE) zur Analyse von Multiproteinkomplexen aus Zelllysaten

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.

Gießen und Ausführen eines Protein-Gel durch die Wiederverwendung von Gewerbe-Kassetten

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

Agarosegel-Elektrophorese für die Trennung von DNA-Fragmenten

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

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.

Mucin Agarose-Gel-Elektrophorese: Western Blotting für Hochmolekulare Glykoproteine


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.

Methoden zur Untersuchung Veränderungen inhärenten Proteinaggregation mit dem Alter 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 ...