Die Autoren danken: Dr. Frank Jiang, Dr. David Lin, Dr. Bernard Yurke und Dr. Uday Chippada für ihre Beiträge zur Entwicklung der DNA-Gel-Technologie; Dr. Norell Hadzimichalis, Smit Shah, Kimberly Peterman, Robert Arter für ihre Kommentare und Bearbeitungen dieser Handschrift; Finanzierungsquellen, einschließlich der New Jersey Kommission auf Rückenmarksforschung (Grant # 07A-019-SCR1, NAL) und New Jersey Neuroscience Institute (MLP); und Herausgeber von Tissue Engineering, Teil A, für die Erlaubnis, 2 und 4 und Biomaterialien für die Erlaubnis, Abbildung 3 Reprint.

<ol>
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Die Autoren haben nichts zu offenbaren.

Die Fähigkeit von DNA-Gelen zu erweichen oder zu versteifen, bevor und nachdem die Zelladhäsion macht sie zu einem idealen Modell, um die Rolle von Gewebesteifigkeit dynamisch auf die Zellfunktion untersucht werden. Alle drei Ausführungen sind in mechanischen und biologischen Untersuchungen verwendet. Allerdings haben alle drei Ausführungen ähnlich Elastizitäten bei verschiedenen Vernetzungsprozent anzeigt Vernetzungslänge nicht beeinflusst DNA Gel-Elastizität (Tabelle 2). Im Gegensatz dazu betr...

Statische und dynamische Substrate gibt zwei Kategorien von Biomaterialien, die entwickelt wurden, um die Wirkungen einer Gewebe Elastizität oder Steifigkeit auf die Zellfunktion untersucht werden. Statische Substrate sind nicht in der Lage, ihre physikalischen Eigenschaften ändern, nachdem sie hergestellt sind, und / oder einmal Zellen plattiert. Polyacrylamid (PA)-Gelen wurden die ersten zweidimensionalen statischen Substraten für Untersuchungen Mechanobiologie 5,17 synthetisiert. PA Gele sind leicht herzustellen, preiswert, flexibel und kann mit einer Vielzahl von elastischen Moduli hergestellt werden. Obwohl diese technischen Vorteile machen PA-Gele häufig angewendet Substrat, sind statische Substrate kein Hinweis auf die Dynamik der extrazellulären Matrix (ECM) und die umliegenden Zellumgebung in vivo. Zum Beispiel kann die ECM-Steifigkeit erfährt Änderungen als Folge von Verletzungen, die Entwicklung oder Krankheit. Dynamische Substrate werden daher als gehirnähnlichen Substrat Modelle in Mechanobiologie Studien begünstigt <sup&gt; 22,24,25. Zahlreiche synthetische, natürliche, zweidimensionalen, dreidimensionalen, statischen und dynamischen Biomaterialien wurden entwickelt, um Gewebesteifigkeit 1,3,6,16,23,26 imitieren. Einige dynamische Substrate benötigen Wärme, UV, elektrischen Strom, Ionen und pH-Änderungen, um ihre mechanischen Eigenschaften zu verändern 2,4,7,8,12,15,16, aber diese Reize Bio-Anwendung des Hydrogels zu beschränken. DNA-Polyacrylamid vernetzt Hydrogele (DNA Gele) sind dynamisch zweidimensionalen elastischen Substraten. DNA-Vernetzungen ermöglichen zeitlichen, räumlichen und reversible Modulation der DNA-Gel-Steifigkeit durch die Zugabe von Einzelstrang-DNA (ssDNA), um Medien oder Puffer 9-11,13,14,18,21. Im Gegensatz zu den oben erwähnten dynamischen Gele wo Stimuli zur Modulation der Elastizität aufgebracht, stützen sich die DNA Gele auf der Diffusion von Applied ssDNA zur Veränderung der Elastizität. Deshalb ist die obere Geloberfläche, wo Zellen gezüchtet werden, ist der erste Bereich moduliert, da die Geschwindigkeit of Elastizität Modulation hängt von der Gel-Dicke. DNA-Gele sind ähnlich wie ihre Gegenstücke in PA-Gel, dass sie eine Polyacrylamid-Rückgrat haben jedoch die Bis-Acrylamid Vernetzungen mit Vernetzungen von DNA (Figur 1) zusammengesetzt ersetzt. Zwei ssDNAs (SA1 und SA2) hybridisieren mit einem Vernetzer Strang (L2) bilden die DNA-Quervernetzungen des Gels. SA1 und SA2 haben verschiedene Sequenzen, die sowohl eine ACRYDIT Modifikation am 5&#39;Ende für eine effektive Einbindung in die PA-Netzwerk enthalten. Zur Herstellung des Gels, SA1 und SA2 werden einzeln in einem PA Gerüst polymerisiert und anschließend die polymerisierten SA1 und SA2 werden miteinander vermischt. L2, das Vernetzungsmittel wird zu der SA1 und SA2 Mischung zugegeben. Der L2-Basensequenz komplementär zu beiden SA1 und SA2 und L2 Sequenzen hybridisiert SA1 und SA2, um die DNA-Vernetzungen zu bilden. Initial, DNA-Gel Elastizität wird von beiden L2-Konzentrationen und Vernetzung (Tabellen 1 bestimmt </strong&gt; und 2). DNA-Gelen, die gleiche stöchiometrische Mengen L2, SA1, SA2 und sind die härtesten Gele, weil SA1 und SA2 sind 100% vernetzt durch L2 (als 100% Gele bezeichnet). Niedrigere Konzentrationen von L2 zu einem niedrigeren Prozentsatz an DNA-Vernetzung und damit weicher DNA Gele. Gele so niedrig wie 50% vernetzt (zu 50% Gele bezeichnet) wurden konstruiert, 9-11. <img alt="Figur 1" src="/files/ftp_upload/51323/51323fig1highres.jpg" /> Abbildung 1. DNA-Gel Vernetzung und uncrosslinking schematische 9-11,13,14,18,21. Schritt 1: SA1 (rot) und SA2 (blau) werden einzeln in einem Polyacrylamid-Rückgrat (schwarz) polymerisiert. Nach der Polymerisation werden SA1 und SA2 polymerisiert Lösungen miteinander vermischt. Schritt 2: L2 (grün) zu und hybridisiert mit SA1 und SA2, die Vernetzungen des Gels. Schritt 3: R2 hybridisiert mit ter Ansatzpunkt von L2. Schritt 4: Brückenkopf-Hybridisierung von R2 treibt das Entpacken von L2 von SA1 und SA2. Im Gegensatz zu PA-Gele können DNA-Gele zu versteifen und zu erweichen nach der Synthese. Aus diesem Grund können die Zellen auf DNA-Gelen gezüchtet, um dynamische Steifigkeit Änderungen unterzogen werden. Zellhaftenden Gelen erstarren kann L2 zu den Kulturmedien von geringen Prozentsatz Gels zugegeben werden, um den Prozentsatz von Vernetzungen zu erhöhen. Erweichen zellhaft Gele können L2 entfernt werden, um den Prozentsatz von Vernetzungen 10,13,21 verringern. L2 hat einen zusätzlichen Brückenkopf-Sequenz am 3&#39;Ende zu ermöglichen L2 von SA1 und SA2 (Tabelle 1) uncrosslink. Entfernung von L2 durch Hybridisierung einer Umkehrstrang genannt R2 erreicht. R2 ist komplementär zu der vollen Länge von L2 und hybridisiert mit dem ersten Ansatzpunkt L2. Brückenkopf-Hybridisierung treibt das Entpacken von L2 von SA1 und SA2, die die Vernetzungs beseitigt und reduziert das Gel Steifigkeit. In diesem Bericht undVideo, Schritt-für-Schritt-Anleitungen für die Herstellung von Versteifungs und Erweichung DNA-Gelen zur Verfügung gestellt. Während 100% und 80% Gel Präparate beschrieben, kann dieses Protokoll zugeschnitten werden, um DNA-Gelen anderer anfänglichen und endgültigen vernetzten Prozentsätze zu erstellen. Im allgemeinen sind 100% und 80% Gele hergestellt, auf Glas-Deckgläschen immobilisiert, funktionalisiert und mit Zellen besät. L2 ist mit den Medien 80% Gele gegeben und R2 ist mit dem Medium 100% Gelen, 48 h nach dem Plattieren zugegeben. Die Zugabe von L2 Medien versteift 80% bis 100% Gelen vernetzt, während die Zugabe von R2 Medien erweicht 100% Gele zu 80% vernetzt ist. Versteift Gele sind als 80 → 100% Gele bezeichnet und aufgeweicht Gele als 100 → 80% Gele im Text bezeichnet. Für die Kontrolle oder statische Gele, ssDNA aus Ts oder As ist, um einen weiteren Satz von 100% und 80% Gelen geliefert. Nach einem Minimum von zwei Tagen nach der Elastizität Modulations können Zellen verarbeitet und analysiert werden. <table border="0" cellpadding="0" cellspacing="0" fo:kEEP-together.within-page = &quot;always&quot;&gt; <tbody align="center"><tr><td rowspan="2"></td><td rowspan="2"> DNA-Vernetzung </td><td rowspan="2"> # Basen </td><td> Sequenz (Brückenkopf) </td><td rowspan="2"> Änderung </td><td rowspan="2"> Schmelztemperatur (T m ° C) </td><td rowspan="2"> Kommentare </td></tr><tr><td> 5 &#39;→ 3&#39; </td></tr><tr><td rowspan="4"> Design 1 </td><td> SA1 </td><td> 10 </td><td> GCA CCT TTG C </td><td> 5 &#39;ACRYDIT </td><td> 34,9 </td><td></td></tr><tr><td> SA2 </td><td> 10 </td><td> GTC AGA ATG A </td><td> 5 &#39;ACRYDIT </td><td> 23,6 </td><td></td></tr><tr><td> L2 </td><td> 30 </td><td> TCA TTC TGA C GC AAA GGT GC G CTA CAC TTG </td><td></td><td> 56 </td><td>Ein 10 bp-Sequenz Ansatzpunkt ist inbegriffen. </td></tr><tr><td> R2 </td><td> 30 </td><td> CAA GTG CGC TAG ACC TTT GCG TCA GAA TGA </td><td></td><td></td><td> R2 ist komplementär zu L2 </td></tr><tr><td rowspan="4"> Design 2 </td><td> SA1 </td><td> 14 </td><td> CGT GGC ATA GGA CT </td><td> 5 &#39;ACRYDIT </td><td> 46,9 </td><td></td></tr><tr><td> SA2 </td><td> 14 </td><td> GTT TCC CAA TCA GA </td><td> 5 &#39;ACRYDIT </td><td> 40,2 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCT GAT TGG GAA AC A GTC CTA TGC CAC G GT TAC CTT CAT C </td><td></td><td> 65,9 </td><td> Ein 12-bp-Sequenz Ansatzpunkt ist inbegriffen. </td></tr><tr><td> R2 </td><td> 40 </td><td> GAT GAA GGT AAC CGT GGC ATA GGA CTG TTT CCC AAT CAG A </td><td></td><td> 65,9 </td><td> R2 ist komplementär zu L2 </td></tr><tr><td rowspan="3"> Design 3 </td><td> SA1 </td><td> 20 </td><td> ACG GAG GTG TAT GCA ATG TC </td><td> 5 &#39;ACRYDIT </td><td> 55 </td><td></td></tr><tr><td> SA2 </td><td> 20 </td><td> CAT GCT TAG GGA CGA CTG GA </td><td> 5 &#39;ACRYDIT </td><td> 56,6 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCC AGT CGT CCC TAA GCA TG G ACA TTG CAT ACA CCT CCG T </td><td></td><td> 68,8 </td><td> Ansatzpunkt ist nicht inbegriffen. </td></tr><tr><td rowspan="2"> Kontrolle </td><td rowspan="2"> Kontrolle </td><td rowspan="2"> 20-40 </td><td> AAA AAA (etc.) oder </td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td> TTT TTT (usw.) </td></tr></tbody></table> Tabelle 1. Basissequenzen für ssDNA 9-11,13,14,18,21. Zelluläre und mechanischen Studien haben mehrere d verwendetifferent Vernetzungs Designs zur DNA-Gelen mit verschiedenen statischen und dynamischen mechanischen Eigenschaften zu erzeugen. Die in der Quer Design moduliert Parameter sind Basensequenz und Sequenzlänge oder vernetzen Länge. Bold und kursiv Schriften veranschaulichen Basenpaarung zwischen SA1 und L2 und zwischen SA2 und L2. <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody align="center"><tr><td rowspan="2"></td><td colspan="8"> Design </td></tr><tr><td colspan="3"> 1 </td><td colspan="3"> 2 </td><td colspan="2"> 3 </td></tr><tr><td> Acrylamidkonzentration (%) </td><td colspan="3"> 10 </td><td colspan="3"> 10 </td><td> 10 </td><td> 4 </td></tr><tr><td> SA1 und SA2 auf den L2-hybridisiert (% vernetzt) </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 100 </td><td> 100 </td></tr><tr><td> &lt;strong&gt; Elastizität (kPa, Mittelwert ± SEM) </td><td> 6,6 ± 0,6 </td><td> 17,1 ± 0,8 </td><td> 29,8 ± 2,5 </td><td> 5,85 ± 0,62 </td><td> 12,67 ± 1,33 </td><td> 22.88 ± 2.77 </td><td> 25,2 ± 0,5 </td><td> 10,4 ± 0,6 </td></tr></tbody></table> Tabelle 2. Elastizitätsmodul (E) von DNA-Gelen 9-11,13,14,18,21. Acrylamidkonzentration, Vernetzungsprozent und Vernetzungs Länge kann in DNA-Gelen moduliert werden. Motive 1, 2 und 3 sind 20, 28 und 40 bp Vernetzungslängen sind. 100%-Gele für alle Ausführungen haben ähnliche Module anzeigt vernetzen Länge hat keinen Einfluss auf Gel-Elastizität. Variationen in Acrylamidkonzentration verändern jedoch DNA-Gel Elastizität.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ssDNA</td>
			<td>Integrated DNA Technologies (Coralville, Iowa) idtdna.com</td>
			<td></td>
			<td>Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix.</td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>100X Tris-EDTA buffer (TE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO) sigmaldrich.com</td>
			<td>T9285</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Borate-EDTA buffer (TBE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>93290</td>
			<td>TBE is a reproductive toxin.</td>
		</tr>
		<tr>
			<td>40% Acrylamide solution</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>BP14021</td>
			<td>Acrylamide is a toxin.</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>A3678</td>
			<td>Prepare a 2% solution in TE buffer. APS is a toxin and irratant.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>T9281</td>
			<td>Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin.</td>
		</tr>
		<tr>
			<td rowspan="2">12-mm diameter round coverglass</td>
			<td rowspan="2">Fisher Scientific (Pittsburg, PA) fishersci.com</td>
			<td rowspan="2">12-545-82</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Norland optical adhesive 72</td>
			<td>Norland Products (Cranbury, NJ) norlandprod.com</td>
			<td>NOA72</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">24-well tissue culture plate</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td rowspan="2">Microcentrifuge tubes</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>ProteoChem or Thermo Fisher, (Rockland, IL) proteochem.com or thermofisher.com</td>
			<td>C111 or 22589</td>
			<td>Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37&#176;C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure.</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>P6407</td>
			<td>Prepare a 0.2 mg/ml solution in water and filter sterilize.</td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Affymetrix (Santa Clara, CA) affymetrix.com</td>
			<td>13813</td>
			<td>Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive.</td>
		</tr>
		<tr>
			<td>22 X 60 cover glass</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive-displacement pipette</td>
			<td>Gilson, Inc (Middletown, WI) gilson.com</td>
			<td>F148504</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light source</td>
			<td></td>
			<td></td>
			<td>Place gels as close as possible to the UV light. UV light can cause skin or eye injury.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>GTS-Welco (Flemington, NJ) www.praxairmidatlantic.com/</td>
			<td>NI 5.0UH-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dna-crosslinked polyacrylamide hydrogels

Mechanobiologie ist ein aufstrebendes Forschungsgebiet, das die kritische Rolle der körperliche Signale in der Leitung der Zellmorphologie und Funktion adressiert. Zum Beispiel ist die Wirkung der Elastizität des Gewebes auf die Zellfunktion ein wichtiger Bereich der Forschung, da Mechanobiologie Gewebesteifigkeit moduliert mit der Krankheit, Entwicklung und Verletzungen. Statische Gewebe-ähnlichen Materialien oder Materialien, die nicht ändern kann, wenn die Steifigkeit Zellen plattiert werden hauptsächlich verwendet, um die Effekte der Gewebesteifigkeit auf die Zellfunktionen zu untersuchen. Während Informationen aus statischen Studien gesammelt wertvoll ist, sind diese Studien kein Hinweis auf die dynamische Natur der zellulären Mikroumgebung in vivo. Um die Wirkungen der dynamischen Steifigkeit auf die Zellfunktion besser erfüllen eine DNA-vernetzte Polyacrylamid-Hydrogel-System (DNS-Gele) entwickelt. Im Gegensatz zu anderen dynamischen Substraten, haben DNA-Gelen die Fähigkeit zu verringern oder zu erhöhen in der Steifigkeit nach der Herstellung ohne Reize. DNA-Gele bestehen aus DNA crosslTinten, die in einem Polyacrylamid-Grundgerüst polymerisiert werden. Hinzufügen und Entfernen von Vernetzungen durch Lieferung von Einzelstrang-DNA kann zeitlicher, räumlicher und reversible Steuerung Gelelastizität. Wir haben in früheren Berichten dargestellt, dass die dynamische Modulation der DNA-Gel Elastizität beeinflusst Fibroblasten und Neuronen Verhalten. In diesem Bericht und Video bieten wir eine schematische, die die DNA-Gel Vernetzungsmechanismen und Schritt-für-Schritt-Anleitung beschreibt auf die Vorbereitung DNA-Gelen.

Vor unserer Studien wurden zell ECM-Wechselwirkungen auf statische konform Biomaterialien oder irreversibel und unidirektionalen dynamischen Substraten beobachtet. Diese Substrate müssen nicht genau die dynamische Natur der zellulären Mikroumgebung. Unsere Arbeit verschiebt die bestehenden technischen Paradigmen, indem sie einen physiologischen Modell zur Untersuchung von Zell-ECM-Interaktionen auf Erweichung und Versteifungs dynamische Biomaterialien. In früheren Studien untersuchten wir die Wirkungen von dynamische...

Watch this Scientific Journal Video about Preparation of DNA-crosslinked Polyacrylamide Hydrogels at JoVE.com

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Unser Labor hat DNA-vernetzte Polyacrylamid-Hydrogele, ein dynamisches System Hydrogel entwickelt, um die Effekte der Modulation Gewebesteifigkeit auf die Zellfunktion zu verstehen. Hier bieten wir Schaltpläne, Beschreibungen und Protokolle, um diese Hydrogele vorzubereiten.

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, ...

preparation-of-dna-crosslinked-polyacrylamide-hydrogels

Research

JoVE Journal

Biology

14.4K Aufrufe.  JFK Medical Center.  Unser Labor hat DNA-vernetzte Polyacrylamid-Hydrogele, ein dynamisches System Hydrogel entwickelt, um die Effekte der Modulation Gewebesteifigkeit auf die Zellfunktion zu verstehen. Hier bieten wir Schaltpläne, Beschreibungen und Protokolle, um diese Hydrogele vorzubereiten. 

Serie: Preparation of DNA-crosslinked Polyacrylamide Hydrogels

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 ...

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 ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Cellular Encapsulation in 3D Hydrogele für Tissue Engineering

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

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 ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Mechanische Stimulation der Chondrozyten-Agarose Hydrogele

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET-Imaging im dreidimensionalen Hydrogelen

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

Proteolytisch abgebaute Alginat-Hydrogele und hydrophobe Mikrobioreaktoren zur Poren-Oozyten-Verkapselung

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Nachweis von Glykosaminoglykanen durch Polyacrylamid-Gelelektrophorese und Silberfärbung

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Ein verbesserter Chemotaxis-Assay zur schnellen Identifizierung von rhizobakteriellen Chemoattraktantien in Wurzelexsudaten

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Einbettung von CFPS-Reaktionen in eine Vielzahl von Hydrogel-Matrizen

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. While the dominant paradigm for the deployment of gene networks is within a cellular chassis, synthetic gene networks may also be deployed in cell-free environments. Promising applications of cell-free gene networks include biosensors, as these devices have been demonstrated against biotic (Ebola, Zika, and SARS-CoV-2 viruses) and abiotic (heavy metals, sulfides, pesticides, and other organic contaminants) targets. Cell-free systems are typically deployed in liquid form within a reaction vessel. Being able to embed such reactions in a physical matrix, however, may facilitate their broader application in a wider set of environments. To this end, methods for embedding cell-free protein synthesis (CFPS) reactions in a variety of hydrogel matrices have been developed. One of the key properties of hydrogels conducive to this work is the high-water reconstitution capacity of hydrogel materials. Additionally, hydrogels possess physical and chemical characteristics that are functionally beneficial. Hydrogels can be freeze-dried for storage and rehydrated for use later. Two step-by-step protocols for the inclusion and assay of CFPS reactions in hydrogels are presented. First, a CFPS system can be incorporated into a hydrogel via rehydration with a cell lysate. The system within the hydrogel can then be induced or expressed constitutively for complete protein expression through the hydrogel. Second, cell lysate can be introduced to a hydrogel at the point of polymerization, and the entire system can be freeze-dried and rehydrated at a later point with an aqueous solution containing the inducer for the expression system encoded within the hydrogel. These methods have the potential to allow for cell-free gene networks that confer sensory capabilities to hydrogel materials, with the potential for deployment beyond the laboratory.

Autor im Rampenlicht: Ein neuartiger Ansatz zur Einbettung zellfreier Proteinsynthesereaktionen in Hydrogele 

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

Methoden zur Einbettung zellfreier Proteinsynthesereaktionen in makroskalige Hydrogele

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...

Methods Article

Herstellung von DNA-vernetztem Polyacrylamid, Hydrogele