Diese Arbeit wurde von der Singapore Academic Research Council (ARC) [Förderkennzeichen 90/07] unterstützt. Wir danken radiodurans für die Unterstützung.

Die vollständige und detaillierte Text-Protokoll für dieses experimentelle Vorgehen ist in Current Protocols in Molecular Biology. Detaillierte Schritt-für-Schritt-Anleitungen für die Montage des Gels Sandwich und für Gel-Apparatur kann auf der Website BioRad 3 gefunden werden. Erforderliche Ausrüstung: Glasplatten (innere und äußere) 10 cm-Zelle: 10,1 x 7,3 cm (Innen-Platte), 10,1 x 8,2 cm (äußere Platte), BioRad 20 cm-Zelle: 20 x 20 cm (innere Platte), 20 x 22,3 cm (äußere Platte), BioRad 0.5-1.5 mm Gel-Kamm und Spacer Gel Gelgießstand Gel Gerät mit Deckel und Kabel Hochspannungs-Netzgerät Heizblock oder Wasserbad Serologische Pipetten und Pipettenspitzen Hilfe Pipette und Pipettenspitzen Gel Trockner oder Scanner Reagenzien und Lösungen: Urea (Reinstwasser) 40% Polyacrylamid-Lösung (29:1) 10 x TBE-Lösung (Tris-Borat, EDTA-Puffer) Deionisiertes, destilliertes Wasser TEMED 30% (w / v) Ammoniumpersulfat-Lösung 0,5 x TBE-Lösung Formamid EDTA Xylencyanol Bromphenolblau Methanol Ethanol <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Volumen </td><td colspan="3" style="text-align:center;"> 50 ml </td><td colspan="3" style="text-align:center;"> 60 ml </td><td colspan="3" style="text-align:center;"> 10 ml </td></tr><tr><td> Acrylamid-Konzentration </td><td> 10% </td><td> 12,5% </td><td> 15% </td><td> 10% </td><td> 12,5% </td><td> 15% </td><td> 10% </td><td> 12,5% </td><td> 15% </td></tr><tr><td> g Harnstoff </td><td> 24 </td><td> 24 </td><td> 24 </td><td> 28,8 </td><td> 28,8 </td><td> 28,8 </td><td> 4,8 </td><td> 4,8 </td><td> 4,8 </td></tr><tr><td> ml 40% Acryl (29:1) </td><td> 12,5 </td><td> 15,625 </td><td> 18,75 </td><td> 15 </td><td> 18,75 </td><td> 22,5 </td><td> 2,5 </td><td> 3,125 </td><td> 3,75 </td></tr><tr><td> ul 30% APS </td><td> 166 </td><td> 166 </td><td> 166 </td><td> 199,2 </td><td> 199,2 </td><td> 199,2 </td><td> 33 </td><td> 33 </td><td> 33 </td></tr><tr><td> ul TEMED </td><td> 20 </td><td> 20 </td><td> 20 </td><td> 24 </td><td> 24 </td><td> 24 </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 10 x TBE </td><td> 5 </td><td> 5 </td><td> 5 </td><td> 6 </td><td> 6 </td><td> 6 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td colspan="10"> Füllen Sie das Volumen mit deionisiertem, destilliertem Wasser </td></tr></tbody></table> Tabelle 1: Reagenzien und Lösungen für die verschiedenen Acrylamid-Konzentrationen und Volumina für die Lösung einzelner Oligonukleotide benötigt Gel-Sandwich Montage und Gelzubereitung <ol><li> Montieren Sie das Gel nach Herstellerangaben Beschreibung und fix das Gel in die Gel-Casting Kammer 3. Verwenden 0,5-1,5 mm dicke Abstandshalter. </li><li> Die geeignete Polyacrylamid-Lösung nach aktuellen Protokolle der Molekularbiologie oder, wie in Tabelle 1 aufgeführt. Für eine Denaturierung Acrylamid-Gel von 20 cm x 22 cm x 1,5 mm, 60 ml Gel-Lösung und für ein 10,1 x 8,2 cm x 1 mm Gel 5 ml Gel-Lösung ist ausreichend. Größere Gele verwendet werden, wenn die erwarteten Produkte / Bands im Bereich von wenigen Basen sind, dann eine längere Gel lösen Bands mit einer Differenz von einem einzigen Nukleotid. Wenn die erwarteten Banden in mehr als 10 Nukleotide unterscheiden, werden kleinere Gele geeignet sein, um die Fragmente zu lösen. Wählen Sie den Prozentsatz der Polyacrylamid, dass Ihre Trennung Anforderungen entspricht, wird eine höhere Polyacrylamid-Konzentrationen lösen niedrigerem Molekulargewicht Fragmente. Verwenden Sie Reinstwasser Harnstoff und vermischen sich mit der gewünschten Menge an Acrylamid. Add TBE-Puffer, um das Gel mischen, um eine endgültige Konzentration von 0,5-1 x TBE bekommen und füllen Sie das Volumen mit deionisiertem, destilliertem Wasser. Erhitzen Sie die Lösung für 20 Sekunden in der Mikrowelle und mischen Sie es sanft. Für größere Gelvolumina wiederholen Sie diesen Schritt, bis die Lösung handwarm. </li><li> Gießen Sie das Gel sofort mit einer serologischen Pipette und einer automatischen Pipette Hilfe zwischen den beiden Glasplatten. Vermeiden Sie die Einführung von Luftblasen. Setzen Sie den Kamm und lassen Sie das Gel für 30-60 Minuten polymerisieren. </li></ol> Richten Sie die Elektrophorese-Apparatur und Vorlauf des Gels <ol><li> Demontieren Sie das Gel aus der Giesskammer und der Montage auf das Gel gemäß produziert Anleitung 3. </li><li> Füllen Sie das untere Pufferkammer wit Laufpuffer (0.5-1 x TBE), dass die Glasplatten werden subzusammengeführt 2-3 cm mit Puffer. Füllen Sie die obere Pufferkammer bis an die Spitze des Gels mit Laufpuffer. </li><li> Entfernen Sie vorsichtig den Kamm und spülen Sie die Taschen mit Laufpuffer mit Hilfe einer Pipette und Ladepuffer Tipps. </li><li> Bringen Sie den Deckel des Gel-System und stecken Sie die Kabel zu einem Hochspannungs-Stromversorgung. Bevor Sie Ihre Proben laden können, müssen Sie das Gel für mindestens 30 Minuten Vorlauf des Gels erhitzen und die verbleibenden Harnstoff aus dem Gel zu entfernen. Die optimale Temperatur sollte zwischen 45-55 ° C. Vermeiden Sie Temperaturen über 60 ° C als Banden konnten Abstrich oder die Glasplatten knacken könnte. Wählen Sie konstante Watt für den Vorlauf (15-25 W pro Gel). </li></ol> Probenvorbereitung <ol><li> In der Zwischenzeit bereiten Sie Ihre Proben. Daher fügen Sie die entsprechende Menge von 2 x Gelbeladung Mix Ihrer Probe. Der Laden-Mix enthält in der Regel 90% Formamid, 0,5% EDTA, 0,1% Xylencyanol und 0,1% Bromphenolblau. </li><li> Bevor die Proben auf das Gel aufgetragen werden kann, müssen Proben werden hitzedenaturiert durch Erhitzen der Proben zwischen 70-90 ° C für ein paar Minuten. </li></ol> Laden und Ausführen des Gels <ol><li> Wenn der Vorlauf beendet ist, entfernen Sie den Deckel und spülen Sie die Taschen gründlich wie vor als Harnstoff hat sich in den Vertiefungen ausgewaschen beschrieben. </li><li> Übernehmen Sie die Proben vorsichtig aus dem Boden der Tasche. Vermeiden Sie die Einführung von Luftblasen. Loading Puffer zu leeren Taschen angewendet werden, um gleiche Bedingungen während des Laufes zu halten. </li><li> Montieren Sie den Deckel, und führen Sie das Gel bei konstanten Watt zu einem Gel Temperatur von 55 ° C zu halten ähnlich dem Vorlauf. Beachten Sie die Migration der Marker-Farbstoffe, bis der Farbstoff vor Erreichen des unteren Endes des Gels. Die Laufzeit beträgt abhängig von den Prozentsatz der verwendeten Acrylamid, Ionenstärke des Puffers und Geldicke. Ein Lauf kann zwischen 2-4 Stunden dauern. Prüfen Sie auf die Temperatur des Gels. Ein Gel-Thermometer kann helfen, die richtige Temperatur während des Laufs zu überwachen. </li></ol> Prozess des Gels <ol><li> Wenn der Farbstoff vor das Ende des Gels erreicht Das Gel wird aus dem unteren Pufferkammer. Entfernen Sie das Gel aus der Kammer und lösen Sie die Klammern. Ziehen Sie die Abstandhalter und vorsichtig verstellen den Glasplatten. Wenn nötig entfernt der oberen und Gel Teil abgeschnitten. </li><li> Sorgfältig Transfer des Gels in eine Schale mit Gel Fixation Lösung (gleiche Konzentration an TBE plus 5-10% Methanol und Ethanol). Lassen Sie das Gel in die Lösung für 5-10 Minuten und ändern Sie den Puffer zweimal. </li><li> Das Gel ist bereit für die weitere Verarbeitung mit einem Staubsauger Geltrockner oder direktes Scannen des Gels. </li></ol>

<ol>
	<li>Maniatis, T., Jeffrey, A., van deSande, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chain+length+determination+of+small+double-+and+single-stranded+DNA+molecules+by+polyacrylamide+gel+electrophoresis.">Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.</a> Biochemistry. 14, 3787-3794 (1975).</li><li>Albright, L. M., Slatko, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+polyacrylamide+gel+electrophoresis.">Denaturing polyacrylamide gel electrophoresis.</a> Curr Protoc Nucleic Acid Chem. , (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PROTEAN II xi Cell and PROTEAN II xi 2-D Cell Instruction Manual. , (2001).</li></ol>

The authors have nothing to disclose.

<ol><li> Die Band Schärfe hängt von mehreren Faktoren ab. Das Volumen der Probe für scharfe Banden geladen werden sollte, so klein wie möglich, also im Idealfall zwischen 1-5 pl. Allerdings können bis zu 15 ul geladen, die noch eine akzeptable Auflösung. Weniger Probenvolumen Ergebnisse in schärfere Banden. </li><li> Die Band Qualität ist auch abhängig von der Gel-Dicke. Thinner Gelen, wie z. B. 0,75 mm zeigen bessere Ergebnisse als 1,5 mm dicken Gele. </li><li> Die Form der Bänder können auch während der Gel-loading beeinflusst wer...

denaturing urea polyacrylamide gel electrophoresis (urea page)

Harnstoff-PAGE oder denaturierende Harnstoff-Polyacrylamid-Gelelektrophorese beschäftigt 6-8 M Harnstoff, der sekundären DNA-oder RNA-Strukturen denaturiert und wird für die Trennung in einem Polyacrylamid-Gel-Matrix auf das Molekulargewicht eingesetzt. Fragmente zwischen 2 bis 500 Basen, mit einer Länge von Differenzen so klein wie ein single nucleotide können getrennt mit dieser Methode 1 sein. Die Migration der Probe ist abhängig von der gewählten Acrylamid-Konzentration. Ein höherer Prozentsatz von Polyacrylamid löst niedrigerem Molekulargewicht Fragmente. Die Kombination von Harnstoff und Temperaturen von 45-55 ° C während der Gel laufen zu lassen für die Trennung von unstrukturierten DNA-oder RNA-Moleküle. Im allgemeinen wird diese Methode ist erforderlich, um zu analysieren oder zu reinigen Einzelstrang-DNA oder RNA-Fragmente, wie synthetisiert oder markierte Oligonukleotide oder Produkte aus der enzymatischen Spaltung Reaktionen. In diesem Video-Artikel zeigen wir, wie die Vorbereitung und Ausführung der denaturierenden Harnstoff-Polyacrylamidgelen. Technische Tipps enthalten sind, zusätzlich zu den Original-Protokoll 1,2.

Watch this Scientific Journal Video about Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE at JoVE.com

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturierung Harnstoff-Polyacrylamid-Gelelektrophorese wird verwendet, um Einzelstrang-DNA oder RNA getrennt bis zu einer Grenze von 500 Nukleotiden. Urea in Kombination mit Hitze denaturiert Proben und unstrukturierte Einzelstränge in der Gel-Matrix migrieren nach ihrem Molekulargewicht.

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 ...

denaturing-urea-polyacrylamide-gel-electrophoresis-urea-page

Research

JoVE Journal

Biology

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

78.9K Aufrufe.  Nanyang Technological University, Singapore - NTU. Denaturierung Harnstoff-Polyacrylamid-Gelelektrophorese wird verwendet, um Einzelstrang-DNA oder RNA getrennt bis zu einer Grenze von 500 Nukleotiden. Urea in Kombination mit Hitze denaturiert Proben und unstrukturierte Einzelstränge in der Gel-Matrix migrieren nach ihrem Molekulargewicht.

Serie: Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

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

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

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

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

Optimierte Fibringel Bead Assay für das Studium der Angiogenese

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

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

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

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 ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Schnell und feinfühlig Kolloidale Coomassie G-250 Färbung für Proteine ​​in Polyacrylamidgelen

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

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 ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Stoffwechselweg der Firmung und der Entdeckung durch 13 C-Kennzeichnung von proteinogenen Aminosäuren

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

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 ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Herstellung von DNA-vernetztem Polyacrylamid, Hydrogele

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

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 ...

Denaturierung Urea Polyacrylamidgelelektrophorese (Urea PAGE)

Zusammenfassung

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

Denaturierung Urea Polyacrylamidgelelektrophorese (Urea PAGE)

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

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

Schnell und feinfühlig Kolloidale Coomassie G-250 Färbung für Proteine ​​in Polyacrylamidgelen

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

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

Schnell und feinfühlig Kolloidale Coomassie G-250 Färbung für Proteine in Polyacrylamidgelen

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