Gli autori desiderano ringraziare: Dr. Frank Jiang, il dottor David Lin, il Dr. Bernard Yurke e il Dr. Uday Chippada per i loro contributi sullo sviluppo della tecnologia del gel del DNA; Dr. Norell Hadzimichalis, Smit Shah, Kimberly Peterman, Robert Arter per i loro commenti e le modifiche di questo manoscritto; fonti di finanziamento, tra cui il New Jersey Commissione su Spinal Cord Research (Grant # 07A-019-SCR1, NAL) e New Jersey Neuroscience Institute (MLP); e editori di Ingegneria Tissutale, parte A, per il permesso di ristampare figure 2 e 4 e Biomateriali per il permesso di ristampare figura 3.

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+rigid+spherical+inclusions+in+young's+moduli+determination:+Application+to+DNA-crosslinked+gels.">Use of rigid spherical inclusions in young's moduli determination: Application to DNA-crosslinked gels.</a> J Biomech Eng. 127 (4), 571-579 (2005).</li><li>Luo, Y., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-activated+immobilization+of+biomolecules+to+agarose+hydrogels+for+controlled+cellular+response.">Light-activated immobilization of biomolecules to agarose hydrogels for controlled cellular response.</a> Biomacromolecules. 5 (6), 2315-2323 (2004).</li><li>Marklein, R. A., Burdick, J. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+in+development+A+growing+role+for+contractility.">Mechanotransduction in development A growing role for contractility.</a> Nat Rev Mol Cell Biol. 10 (1), 34-43 (2009).</li><li>Wuerfel, J., 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=Mr-elastography+reveals+degradation+of+tissue+integrity+in+multiple+sclerosis.">Mr-elastography reveals degradation of tissue integrity in multiple sclerosis.</a> Neuroimage. 49 (3), 2520-2525 (2012).</li><li>Zaari, N., Rajagopalan, P., Kim, S. K., Engler, A. J., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photopolymerization+in+microfluidic+gradient+generators+Microscale+control+of+substrate+compliance+to+manipulate+cell+response.">Photopolymerization in microfluidic gradient generators Microscale control of substrate compliance to manipulate cell response.</a> Adv Mater. 16 (23-24), 2133-2137 (2004).</li></ol>

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La capacità del gel di DNA per ammorbidire o irrigidire prima e dopo l&#39;adesione cellulare li rende un modello ideale per studiare il ruolo della rigidità dei tessuti dinamica sulla funzione delle cellule. Tutti e tre i disegni sono stati utilizzati in studi meccanici e biologici. Tuttavia, tutti e tre i disegni hanno elasticità simili in varie percentuali di reticolazione, indicante la lunghezza di reticolazione non influenza l&#39;elasticità del gel del DNA (Tabella 2). Al contrario, la concent...

Supporti statici e dinamici sono due categorie di biomateriali che sono stati sviluppati per studiare gli effetti di elasticità dei tessuti o di rigidità sulla funzione cellulare. Supporti statici sono in grado di cambiare le loro proprietà fisiche dopo che sono stati fabbricati e / o una volta che le cellule sono placcati. Poliacrilammide (PA) gel sono stati i primi bidimensionali, supporti statici che sono stati sintetizzati per le indagini mechanobiology 5,17. Gel PA sono facili da preparare, economico, versatile e può essere fabbricato con una vasta gamma di moduli elastici. Anche se questi vantaggi tecnici rendono PA gelifica un substrato comunemente applicato, supporti statici non sono indicativi della natura dinamica della matrice extracellulare (ECM) e di ambiente cellulare circostante in vivo. Ad esempio, l&#39;ECM subisce alterazioni rigidità come risultato di lesioni, sviluppo, o malattia. Supporti dinamici sono quindi favoriti come modelli substrato di tessuto-mimando negli studi mechanobiology <sup&gt; 22,24,25. Numerosi sintetico, bidimensionale biomateriali, tridimensionali, statiche e dinamiche naturali sono stati sviluppati per imitare il tessuto rigidità 1,3,6,16,23,26. Alcuni supporti dinamici richiedono calore, ai raggi UV, corrente elettrica, gli ioni, e variazioni di pH per alterare le loro proprietà meccaniche 2,4,7,8,12,15,16, ma questi stimoli possono limitare bio-applicazione del idrogel. Idrogel di poliacrilamide DNA-reticolato (gel di DNA) sono substrati elastici bidimensionali dinamici. Legami crociati DNA permettono la modulazione temporale, spaziale, e reversibile di gel DNA rigidità con l&#39;aggiunta di DNA a singola elica (ssDNA) a supporto o tampone 9-11,13,14,18,21. A differenza dei gel dinamici suddetti in cui si applicano stimoli per la modulazione di elasticità, i gel di DNA basano sulla diffusione di ssDNA applicata per la modifica di elasticità. Pertanto, la superficie del gel superiore, dove si coltivano cellule, è la prima zona modulato perché il tasso omodulazione elasticità f dipende dallo spessore del gel. Gel di DNA sono simili alle loro controparti gel PA dal fatto che essi hanno un backbone poliacrilammide, tuttavia i legami crociati bis-acrilamide vengono sostituiti con legami crociati costituite da DNA (Figura 1). Due ssDNAs (SA1 e SA2) ibridano con un filamento reticolante (L2) per compensare i legami crociati DNA del gel. SA1 e SA2 hanno sequenze distinte che contengono sia una modifica Acrydite alla fine 5&#39;per incorporazione efficace nella rete PA. Per la preparazione del gel, SA1 e SA2 sono individualmente polimerizzati in una spina dorsale PA e, in seguito, la SA1 e SA2 polimerizzato sono mescolati insieme. L2, il reticolante, viene aggiunto alla miscela SA1 e SA2. La sequenza di basi L2 è complementare ad entrambe le sequenze SA1 e SA2 e L2 si ibrida con SA1 SA2 più per formare i legami crociati DNA. , Elasticità del gel DNA iniziale è determinato da entrambe le concentrazioni L2 e reticolazione (tabelle 1 </strong&gt; e 2). Gel di DNA contenenti uguali quantità stechiometriche di L2, SA1, SA2 e sono i gel più rigide perché SA1 e SA2 sono reticolati al 100% da L2 (designato come 100% gel). Concentrazioni minori di risulta L2 in una percentuale inferiore di reticolazione DNA e, quindi, più morbide gel di DNA. Gel a partire da 50% reticolato (designato come il 50% gel) sono stati costruiti 9-11. <img alt="Figura 1" src="/files/ftp_upload/51323/51323fig1highres.jpg" /> Figura 1 gel DNA reticolazione e uncrosslinking schematica 9-11,13,14,18,21 Passo 1:. SA1 (rosso) e SA2 (blu) sono individualmente polimerizzati in una spina dorsale poliacrilammide (nero). Dopo la polimerizzazione, soluzioni polimerizzati SA1 e SA2 sono mescolati insieme. Fase 2: L2 (verde) si aggiunge e si ibrida con SA1 SA2 più per formare i legami crociati del gel. Fase 3: R2 ibrida con tegli toehold di L2. Fase 4: ibridazione Toehold R2 spinge la decompressione di L2 da SA1 e SA2. A differenza dei gel PA, gel DNA possono irrigidirsi e ammorbidire dopo la sintesi. Per questo motivo, le cellule coltivate su gel di DNA possono essere sottoposti a modifiche di rigidità dinamica. Per irrigidire gel cellule aderenti, L2 può essere aggiunto al mezzo di coltura di gel basse percentuali di aumentare la percentuale di legami crociati. Per ammorbidire gel cellule aderenti, L2 può essere rimosso per diminuire la percentuale di legami crociati 10,13,21. L2 ha una sequenza appiglio supplementare al fine di consentire 3&#39;L2 a uncrosslink da SA1 e SA2 (Tabella 1). Rimozione di L2 è compiuta mediante ibridazione di un filamento inversione chiamato R2. R2 è complementare alla lunghezza della L2 e si ibrida con il primo appiglio L2. Toehold ibridazione spinge la decompressione di L2 da SA1 e SA2, che elimina la reticolazione e riduce la rigidità gel. In questa relazione evideo, passo-passo le istruzioni sono fornite per la preparazione di irrigidimento e ammorbidire gel di DNA. Mentre 100% e 80% preparazioni gel sono descritti, questo protocollo può essere adattato per creare gel di DNA di altre percentuali reticolato iniziali e finali. In generale, 100% e 80% gel vengono preparati, immobilizzati su vetrini di vetro, funzionalizzati, e seminati con cellule. L2 viene aggiunto ai media di 80% gel e R2 si aggiunge ai media di 100% gel, 48 ore dopo la placcatura. L&#39;aggiunta di L2 ai media irrigidisce 80% gel 100% reticolato, mentre l&#39;aggiunta di R2 ai media ammorbidisce 100% gel al 80% reticolato. Gel irrigiditi sono designati come 80 → 100% gel e gel ammorbiditi sono designati come 100 → 80% gel nel testo. Per il controllo o gel statici, ssDNA composto da Ts o AS è consegnato ad un altro gruppo di 100% e 80% gel. Dopo un minimo di due giorni modulazione elasticità, le cellule possono essere elaborati ed analizzati. <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"> Reticolazione DNA </td><td rowspan="2"> # Di basi </td><td> Sequence (Toehold) </td><td rowspan="2"> Modifica </td><td rowspan="2"> Temperatura di fusione (T m, ° C) </td><td rowspan="2"> Commenti </td></tr><tr><td> 5 &#39;→ 3&#39; </td></tr><tr><td rowspan="4"> Disegno 1 </td><td> SA1 </td><td> 10 </td><td> GCA CCT TTG C </td><td> 5 &#39;Acrydite </td><td> 34.9 </td><td></td></tr><tr><td> SA2 </td><td> 10 </td><td> GTC AGA ATG A </td><td> 5 &#39;Acrydite </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>Una sequenza toehold 10 bp è incluso. </td></tr><tr><td> R2 </td><td> 30 </td><td> CAA GTG TAG CGC ACC TTT GCG TCA GAA TGA </td><td></td><td></td><td> R2 è complementare a 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;Acrydite </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;Acrydite </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> A 12 bp sequenza appiglio è incluso. </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 è complementare a 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;Acrydite </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;Acrydite </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> Toehold non è incluso. </td></tr><tr><td rowspan="2"> Controllo </td><td rowspan="2"> Controllo </td><td rowspan="2"> 20-40 </td><td> AAA AAA (ecc) o </td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td> TTT TTT (etc.) </td></tr></tbody></table> Tabella 1. sequenze di base per ssDNA 9-11,13,14,18,21. Cellulare e studi meccanici hanno utilizzato diversi ddisegni di reticolazione ifferent per generare gel di DNA con una gamma di statici e dinamici proprietà meccaniche. I parametri modulati nel design di reticolazione sono sequenza di basi e lunghezza della sequenza o la lunghezza di reticolazione. Caratteri in grassetto e in corsivo illustrano appaiamento delle basi tra SA1 e L2 e tra SA2 e L2 rispettivamente. <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> Acrilamide Concentrazione (%) </td><td colspan="3"> 10 </td><td colspan="3"> 10 </td><td> 10 </td><td> 4 </td></tr><tr><td> SA1 SA2 più ibridato a L2 (% reticolato) </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; Elasticità (kPa, media ± 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> Tabella 2 Modulo di Young (E) del gel del DNA 9-11,13,14,18,21. Concentrazione di acrilamide, percentuale di reticolazione, e la lunghezza di reticolazione può essere modulata in gel di DNA. Designs 1, 2, e 3 hanno 20, 28, e 40 bp lunghezze di reticolazione, rispettivamente. 100% gel per tutti i disegni hanno moduli simili indicante la lunghezza di reticolazione non influenza l&#39;elasticità del gel. Tuttavia, le variazioni della concentrazione di acrilamide alterano l&#39;elasticità del gel del DNA.

<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

Mechanobiology è un settore scientifico emergente che affronta il ruolo critico di segnali fisici nel dirigere la morfologia e la funzione delle cellule. Ad esempio, l&#39;effetto di elasticità dei tessuti sulla funzione delle cellule è un importante area di ricerca mechanobiology perché la rigidità del tessuto modula con la malattia, lo sviluppo, e lesioni. Materiali tessuto-mimando statiche, o materiali che non possono alterare la rigidezza volta che le cellule sono placcati, vengono prevalentemente utilizzati per studiare gli effetti della rigidità dei tessuti sulle funzioni cellulari. Mentre le informazioni raccolte da studi statici è prezioso, questi studi non sono indicativi della natura dinamica del microambiente cellulare in vivo. Per affrontare meglio gli effetti della rigidità dinamica sulla funzione delle cellule, abbiamo sviluppato un sistema di poliacrilammide idrogel DNA-reticolato (gel di DNA). A differenza di altri supporti dinamici, gel di DNA hanno la capacità di aumentare o diminuire la rigidità dopo la fabbricazione, senza stimoli. Gel di DNA sono costituiti da DNA crosslinchiostri che vengono polimerizzate in una spina dorsale di poliacrilamide. Aggiunta e rimozione di legami crociati con la consegna di DNA a singolo filamento permette temporale, spaziale, e il controllo reversibile di elasticità del gel. Abbiamo dimostrato in precedenti relazioni che la modulazione dinamica di elasticità del gel DNA influenza dei fibroblasti e neuroni comportamento. In questo rapporto e video, forniamo uno schema che descrive i meccanismi di reticolazione gel del DNA e passo-passo le istruzioni su gel di preparazione del DNA.

Prima i nostri studi, le interazioni cellula-ECM sono stati osservati sui biomateriali compatibili statici o su supporti dinamici irreversibili e unidirezionali. Questi substrati non riflettono con precisione la natura dinamica del microambiente cellulare. Il nostro lavoro si sposta i paradigmi tecnici esistenti, fornendo un modello più fisiologica per lo studio delle interazioni cellula-ECM sul rammollimento e irrigidimento biomateriali dinamici. In studi precedenti, abbiamo analizzato gli effetti dei supporti dinamic...

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

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Il nostro laboratorio ha sviluppato idrogel di poliacrilamide DNA-reticolato, un sistema dinamico di idrogel, per comprendere meglio gli effetti di modulazione rigidità dei tessuti sulla funzione delle cellule. Qui, forniamo schemi, descrizioni e protocolli per preparare questi idrogel.

Preparazione di DNA-reticolato poliacrilammide idrogel

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 Views.  JFK Medical Center.  Il nostro laboratorio ha sviluppato idrogel di poliacrilamide DNA-reticolato, un sistema dinamico di idrogel, per comprendere meglio gli effetti di modulazione rigidità dei tessuti sulla funzione delle cellule. Qui, forniamo schemi, descrizioni e protocolli per preparare questi idrogel. 

Video: 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.

Veloce e sensibile colloidale Coomassie G-250 colorazione per le proteine ​​in gel di poliacrilammide

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

Ricerca

Biologia

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.

Denaturazione Urea elettroforesi su gel poliacrilammide (PAG Urea)

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.

Incapsulamento cellulare in idrogel 3D per l&#39;ingegneria tissutale

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.

Blu nativi poliacrilammide gel elettroforesi (BN-PAGE) per l&#39;analisi di complessi multiproteici da lisati cellulari

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.

La stimolazione meccanica di condrociti-agarosio idrogel

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 in idrogel tridimensionali

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.

Idrogel alginato degradati proteolylylylyd e microbioreattori idrofobici per l'incapsulamento dell'Oocito Porcine

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.

Rilevazione di glicosaminoglicani mediante elettroforesi su gel di poliacrilammide e colorazione dell'argento

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.

Un test di chemiotassi migliorato per la rapida identificazione dei chemioattrattivi rizobatterici negli essudati radicali

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

Incorporamento di reazioni CFPS in una varietà di matrici di idrogel

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.

Autore in primo piano: un nuovo approccio per l'incorporazione di reazioni di sintesi proteica cell-free negli idrogel 

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.

Metodi per incorporare reazioni di sintesi proteica cellulo-free in idrogel su macroscala

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