Grazie al Sig Jason Adkins per l&#39;assistenza tecnica di esperti.

1. sgrassante commerciale gliadina <ol><li> Misurare 150 ml di acetone con un cilindro graduato e versare in 250 ml beuta. <ol><li> Mentre agitazione con una barra di rotazione su un agitatore magnetico a RT, aggiungere 30 g di polvere di gliadina commerciale. Sigillare l&#39;apertura del pallone con un foglio di alluminio, e continuare a mescolare O / N nel cofano. </li></ol></li><li> Filtrare la soluzione con un filtro di carta. <ol><li> Lavare il filtrato con acetone fresco (circa 50 ml). Lasciate riposare per 10 minuti per consentire l&#39;acetone per drenare. </li><li> Trasferire il filtrato insieme con la carta da filtro sotto per un grande piatto, come un quadrato piatto di coltura cellulare. Coprire le stoviglie con una grande carta da filtro per rallentare l&#39;evaporazione di acetone residuo. </li><li> Lasciare che il filtrato si asciughi completamente nella cappa O / N. Conservare la gliadina sgrassata in un contenitore a tenuta d&#39;aria a temperatura ambiente. </li></ol></li></ol> 2. Preparazione di purificata gliadina <ol><li> Misurare 150 ml di WAter con un cilindro graduato e versare in 1.000 ml beuta. <ol><li> Misurare 350 ml di etanolo assoluto con un cilindro disordine e versare nella stessa 1.000 ml beuta. Mescolare energicamente (800 - 1000 rpm) con una barra di rotazione fino nessuna bolla è generata dalla miscela soluzione. </li></ol></li><li> Mentre mescolando, aggiungere 20 g di polvere di gliadina sgrassata commerciale. Aggiungere gliadina in piccole quantità alla volta per evitare la formazione di grumi che contiene aria nel mezzo. Continuate a mescolare O / N. </li><li> Trasferire la soluzione ad un cilindro disordine 1.000 ml. Lasciar riposare per due giorni. Durante questo tempo, le impurità vengono precipitati sul fondo del cilindro. </li><li> Trasferire il supernatante in un evaporatore rotante con una pipetta e rimuovere l&#39;etanolo dal supernatante il più possibile. Come la percentuale di etanolo è ridotto, gliadina apparirà nella soluzione come aggregato. </li><li> Congelare la soluzione contenente aggregati gliadina immergendo / rotazione in metanolo/ Miscela di ghiaccio secco e congelare-secco a -70 ° C sotto vuoto. Prima di liofilizzazione, assicurarsi che l&#39;intera soluzione è congelato, senza alcun liquido. NOTA: La gliadina liofilizzato formerà un solido poroso. </li><li> Crush the gliadina liofilizzato con mortaio e pestello, e macinare con un macinino da caffè per ottenere una multa di potenza (&lt;0.5 mm). Trasferire il prodotto in un contenitore a tenuta d&#39;aria e conservare a temperatura ambiente. </li></ol> 3. polimerizzazione Reazione di ECA con gliadina <ol><li> Mettere una fiala di scintillazione (volume è di circa 20 ml) in un peso di equilibrio, e tara. Aggiungere 3,2 g di acqua distillata e 6,8 g di etanolo assoluto nel flacone scitillation. </li><li> Spostare la fiala di scintillazione su un agitatore magnetico, mettere una barra di rotazione magnetica (20 x 3 mm) nel flaconcino, e mescolare energicamente (800 - 1.000 giri al minuto) fino a quando non bolla d&#39;aria è generato dalla miscela di etanolo acquoso. </li><li> Aggiungere 40 ml di 4 N HCl nel flaconcino mescolando. Aggiungere 20 mg di gliadina nel flacone mentre sti<html> Rring, e continuare a mescolare fino a quando la polvere gliadina è completamente sciolto nella miscela etanolo acquoso. </li><li> Dopo essersi assicurati la soluzione gliadina è chiaro agli occhi nudi, aggiungere lentamente 80 - 100 ml di ECA mentre la velocità di agitazione è ancora 800 - 1.000 giri al minuto. <ol><li> Ridurre la velocità di agitazione a 500 rpm, e continua agitazione per 1 ora. Con il procedere della reazione, osservare torbidità che indica che la reazione di polimerizzazione è in corso. </li></ol></li><li> Quando la reazione è terminata, trasferire la soluzione in provette da centrifuga, e bilanciare il peso dei tubi. Centrifugare il prodotto di reazione a 10.000 xg per 20 min. Durante questo tempo, precipitati di prodotti collaterali e nanoparticelle rimangono nel surnatante. Il componente principale del prodotto lato è PACA omopolimero. </li><li> Dopo la centrifuga, trasferire la sospensione nanoparticelle prodotte (surnatante) con una pipetta ad una fiala di scintillazione (o qualsiasi contenitore ermetico), e conservarlo a RT. </li></ol>title &quot;&gt; 4. caratterizzazione del prodotto <ol><li> Preparare 10 g di etanolo acquoso 68% in peso di acqua mescolando 3,2 g e 6,8 g di etanolo assoluto in 20 ml di scintillazione flacone e agitare fino nessuna bolla è generata dalla miscela soluzione. <ol><li> Aggiungere 40 ml di 4 N HCl alla miscela una soluzione sotto agitazione. Aggiungere 50 ml di sospensione di nanoparticelle alla soluzione di etanolo preparata mescolando. </li></ol></li><li> Misurare la dimensione delle nanoparticelle con Dynamic Light Scattering (DLS) utilizzando la soluzione di campione preparato sopra e seguire le istruzioni del produttore. NOTA: La dimensione del prodotto finale deve essere inferiore a 200 nm. Se è maggiore di 200 nm, il prodotto non sarà stabile per un lungo periodo di tempo. nanoparticelle più grandi sono prodotte quando il lavoratore ECA non è fresco. </li></ol> 5. Esame del Prodotto <ol><li> Preparare una lastra di vetro come uno specchio a mano. Lavare la superficie della lastra di vetro con acqua e sapone. RInse la superficie della lastra di vetro con l&#39;acqua che scorre. Essiccare la superficie pulita o utilizzare senza asciugare per il passaggio successivo. </li><li> Spruzzare la sospensione nanoparticelle ottenuto in 3.6) su una parte della lastra di vetro, e risciacquare la superficie con scorre acqua immediatamente. </li><li> Nebulizzare acqua pura sulla superficie della lastra di vetro. Osservare la (spruzzato con sospensioni di nanoparticelle) strato di acque di superficie rivestita attesa mentre la superficie patinata è chiara. </li></ol>

<ol>
	<li>Vauthier, C., Dubernet, C., Fattal, E., Pinto-Alphandary, H., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(alkylcyanoacrylates)+as+biodegradable+materials+for+biomedical+applications.">Poly(alkylcyanoacrylates) as biodegradable materials for biomedical applications.</a> Adv. Drug Deliver. Rev. 55, 519-548 (2003).</li><li>Wieser, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+of+gluten+proteins.">Chemistry of gluten proteins.</a> Food Microbiol. 24, 115-119 (2007).</li><li>Bietz, J. A., Wall, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity+of+high+molecular+weight+gliadin+and+ethanol+soluble+glutenin+subunits+of+wheat:+Relation+to+gluten+structure.">Identity of high molecular weight gliadin and ethanol soluble glutenin subunits of wheat: Relation to gluten structure.</a> Cereal Chem. 57, 415-421 (1980).</li><li>Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+composites+by+using+gliadin+as+a+bonding+material.">Production of composites by using gliadin as a bonding material.</a> J. Cereal Sci. 54, 168-172 (2011).</li><li>Kim, S., Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+gliadin-poly(ethyl+cyanoacrylate)+nanoparticles+for+hydrophilic+coating.">Production of gliadin-poly(ethyl cyanoacrylate) nanoparticles for hydrophilic coating.</a> J. Nanopart. Res. 16, 1-10 (2014).</li><li>Behan, N., Birkinshaw, C., Clarke, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly+n-butyl+cyanoacrylate+nanoparticles:+a+mechanistic+study+of+polymerisation+and+particle+formation.">Poly n-butyl cyanoacrylate nanoparticles: a mechanistic study of polymerisation and particle formation.</a> Biomaterials. 22, 1335-1344 (2001).</li><li>Nicolas, J., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+poly(alkyl+cyanoacrylate)-based+colloidal+nanomedicines.">Synthesis of poly(alkyl cyanoacrylate)-based colloidal nanomedicines.</a> Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1, 111-127 (2009).</li><li>Kim, S., Evans, K., Biswas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+BSA-poly(ethyl+cyanoacrylate)+nanoparticles+as+a+coating+material+that+improves+wetting+property.">Production of BSA-poly(ethyl cyanoacrylate) nanoparticles as a coating material that improves wetting property.</a> Colloid Surface. B. 107, 68-75 (2013).</li><li>Lander, L. M., Siewierski, L. M., Brittain, W. J., Vogler, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+comparison+of+contact+angle+methods.">A systematic comparison of contact angle methods.</a> Langmuir. 9, 2237-2239 (1993).</li><li>Davies, J., Nunnerley, C. S., Brisley, A. C., Edwards, J. C., Finlayson, S. D. <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+Dynamic+Contact+Angle+Profile+Analysis+in+Studying+the+Kinetics+of+Protein+Removal+from+Steel+Glass,+Polytetrafluoroethylene,+Polypropylene,+Ethylenepropylene+Rubber,+and+Silicone+Surfaces.">Use of Dynamic Contact Angle Profile Analysis in Studying the Kinetics of Protein Removal from Steel Glass, Polytetrafluoroethylene, Polypropylene, Ethylenepropylene Rubber, and Silicone Surfaces.</a> J. Colloid Interf. Sci. 182, 437-443 (1996).</li><li>Giolando, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-crystals+of+titanium+dioxide+in+aluminum+oxide:+A+transparent+self-cleaning+coating+applicable+to+solar+energy.">Nano-crystals of titanium dioxide in aluminum oxide: A transparent self-cleaning coating applicable to solar energy.</a> Sol. Energy. 97, 195-199 (2013).</li></ol>

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

There are several critical steps in the production of the nanoparticle suspension. If the purified gliadin contains impurities, the reaction with ECA will produce side products. Although these unwanted products can be removed from the reaction medium during the centrifugation stage, it lowers the yield of the major product. If the gliadin solution prepared during experimental step 2.3) does not show clear separation between supernatant and precipitate after two days, the solution needs to stand for longer time. Using fre...

The goal of this article is to show the protocol for the preparation of nanoparticle suspension that modifies the wetting property of materials by a simple spray. The presented nanoparticle suspension is made from alkyl cyanoacrylate1 and a cereal protein, gliadin2,3. During the manufacturing process, protein aggregates are formed in aqueous ethanol4. Subsequent reaction with monomer (alkyl cyanoacrylate) produces the nanoparticle that is comprised of a protein core surrounded by linear polymer chains [poly(alkyl cyanoacrylate)]5.
Poly(alkyl cyanoacrylate)s are biodegradable and have been used for the production of nanoparticles via emulsion polymerization6. This reaction is spontaneously initiated by the hydroxyl groups dissociated from water or by other nucleophilic groups in the reaction medium7. In the case of the reaction presented in this article, the amine groups on the surface of protein aggregates initiate the polymerization reaction of alkyl cyanoacrylate monomers5,8. As a result of this reaction, nanoparticles are formed in the reaction medium. The core of the nanoparticle is protein aggregates and the outer layer is poly(alkyl cyanoacrylate) (PACA) chains. The prepared nanoparticle has a strong affinity on most materials (more precisely, any material which PACA can adsorb to) and adheres onto their surface to form a thin coating on a nanometer scale. A simple spray coating instantly turns the surface of the materials hydrophilic.
Gliadin is one of the main fractions of gluten, which is in the endosperms of wheat. Gliadins are mainly monomeric proteins with molecular weights around 28,000 - 55,000. Non-covalent bonds such as hydrogen bonds, ionic bonds and hydrophobic bonds are responsible for the aggregation of gliadins2. Although gliadin is chosen as a reactant in this article, many other proteins can also be used for the same purpose. However, the reaction condition needs to be modified accordingly because the condition for inducing aggregation is dependent on the type of protein to be employed8. Compared with other proteins, gliadin is more readily available, purification is simple, and production cost is low. Although ethyl cyanoacrylate (ECA) is chosen as a monomer for the presented reaction, other alkyl cyanoacrylates can also be used for the same reaction. The reason for choosing ECA is that it is readily available at low cost.

<table border="0" cellpadding="0" cellspacing="0" width="580">
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:143px;">Ethyl cyanoacrylate (ECA) monomer</td>
			<td style="width:113px;">K&#38;R International (Laguna Niguel, CA)</td>
			<td style="width:107px;">I-1605</td>
			<td style="width:219px;">Any pure ECA can be used.</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:143px;">Gliadin</td>
			<td style="width:113px;">MGP Ingredients, Inc (Atchison, KS)</td>
			<td style="width:107px;">Gift from the company</td>
			<td style="width:219px;">Gliadin can be purchased from Sigma-Aldrich (cat #: G3375-25G). Instead of gliadin, any commercial&#160; gluten can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">HCl</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any reagent grade chemical can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Acetone</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any reagent grade chemical can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Methanol</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any reagent grade chemical can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Ethanol (100%)</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any reagent grade chemical can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Filter paper</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any grade filter paper larger than 10 cm can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Cell culture square dish</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any dish larger than 20 cm x 20 cm can be used.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:143px;">Coffee grinder</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any coffee grinder can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Rotary evaporator</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any rotary evaporator can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Freeze Dryer</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any freeze dryer that can reach - 70&#176;C can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Centrifuge</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any centrifuge that can apply 1000 x g can be used.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:143px;">Magnetic stirrer</td>
			<td style="width:113px;">Any</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any magnetic stirrer that can turn spin bar to 1000 RPM can be used.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:143px;">Dynamic Light Scattering (DLS)</td>
			<td style="width:113px;">Brookhaven Instruments Corporation</td>
			<td style="width:107px;">NanoBrook Omni Zeta Potential Analyzer</td>
			<td style="width:219px;">DLS from any company can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Scanning Electron Microscope (SEM)</td>
			<td style="width:113px;">Carl Zeiss Inc.</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any SEM can be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:143px;">Dynamic Contact Angle (DCA)</td>
			<td style="width:113px;">Thermo Cahn Instruments</td>
			<td style="width:107px;"></td>
			<td style="width:219px;">Any DCA can be used.</td>
		</tr>
	</tbody>
</table>

a protocol for the production of gliadin-cyanoacrylate nanoparticles for hydrophilic coating

Questo articolo presenta un protocollo per la produzione di nanoparticelle a base di proteine ​​che cambia la superficie idrofobica ad idrofilica da un semplice rivestimento a spruzzo. Queste nanoparticelle sono prodotte dalla reazione di polimerizzazione di alchil cianoacrilato sulla superficie della proteina cereali (gliadina) molecole. Alchil cianoacrilato è un monomero che polimerizza immediatamente a RT quando viene applicato alla superficie dei materiali. La reazione di polimerizzazione viene iniziata dalle tracce di specie debolmente base o nucleofili sulla superficie, comprese umidità. Una volta polimerizzato, i cianoacrilati alchil polimerizzati mostrano una forte affinità con i materiali degli oggetti, perché i gruppi di nitrile sono la spina dorsale di poli (cianoacrilato alchil). Proteine ​​funzionano anche come iniziatore di polimerizzazione per questo perché contengono gruppi amminici che possono avviare la polimerizzazione di cianoacrilato. Se proteine ​​aggregate viene usato come iniziatore, aggregata proteina è circondato dal idrofobicapoli (alchil cianoacrilato) catene dopo la reazione di polimerizzazione di alchil cianoacrilato. Controllando la condizione sperimentale, particelle nell&#39;intervallo nanometri sono prodotti. Le nanoparticelle prodotte facilmente adsorbono alla superficie della maggior parte dei materiali tra cui vetro, metalli, plastica, legno, pelle e tessuti. Quando la superficie di un materiale è spruzzato con la sospensione nanoparticelle prodotte e risciacquata con acqua, la struttura micellare di nanoparticelle cambia la sua conformazione, e le proteine ​​idrofile sono esposti all&#39;aria. Come risultato, la superficie delle nanoparticelle rivestite cambia in idrofila.

Le nanoparticelle possono essere preparate in varie condizioni di reazione. Forme gliadina aggregano in un&#39;ampia gamma di contenuto di etanolo 5. Tuttavia, la dimensione degli aggregati deve essere il più piccolo possibile, perché un ulteriore strato (es., Polimerizzato ECA) verrà aggiunto a questo aggregato e questo processo renderà la dimensione finale grande. Se la dimensione finale delle particelle è troppo grande, la particella sarà instabile e verrà f...

Watch this Scientific Journal Video about A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating at JoVE.com

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

Un protocollo per la produzione di nanoparticelle gliadina-cianoacrilato per rivestimento idrofilo

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

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6.8K Views.  USDA/ARS/NCAUR. This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

Video: A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Produzione di disolfuro-stabilizzati peptide transmembrana per gli studi strutturali

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

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Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth.&#160;The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions.&#160;This paper describes standard protocols for TLC and contact bioautography&#160;with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light.&#160;Zones are cut out and incubated face down onto agar inoculated with the test microorganism.&#160;Inhibitory bands are visualized by staining the agar plates with tetrazolium red.&#160;The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen.&#160;The TLC methods apply to many types of plant extracts&#160;and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper&#160;ammonia-producing bacteria (HAB) native to the bovine rumen.

Strato sottile cromatografica (TLC) Separazioni e Bioassays di estratti vegetali per identificare composti antimicrobici

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

Un metodo semplice per la dimensione sintesi controllata di stabili oligomeriche Grappoli di oro nanoparticelle in condizioni ambientali

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

Protocollo per la sintesi di<em&gt; Ortho</em&gt; Derivati ​​all&#39;anilina -trifluoromethoxylated

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

A Simple and Efficient Protocol for the Catalytic Insertion Polymerization of Functional Norbornenes

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic nature of the monomer is conserved. The resulting polymer, polynorbornene, has a very high glass transition temperature, Tg, and interesting optical and electrical properties. However, the polymerization of functional norbornenes by this mechanism is complicated by the fact that the endo substituted norbornene monomer has, in general, a very low reactivity. Furthermore, the separation of the endo substituted monomer from the exo monomer is a tedious task. Here, we present a simple protocol for the polymerization of substituted norbornenes (endo:exo ca. 80:20) bearing either a carboxylic acid or a pendant double bond. The process does not require that both isomers be separated, and proceeds with low catalyst loadings (0.01 to 0.02 mol%). The polymer bearing pendant double bonds can be further transformed in high yield, to afford a polymer bearing pendant epoxy groups. These simple procedures can be applied to prepare polynorbornenes with a variety of functional groups, such as esters, alcohols, imides, double bonds, carboxylic acids, bromo-alkyls, aldehydes and anhydrides.

We describe the catalytic insertion polymerization of 5-norbornene-2-carboxylic acid and 5-vinyl-2-norbornene to form functional polymers with a very high glass transition temperature.

Un protocollo semplice ed efficiente per l&#39;inserimento catalitica polimerizzazione di norborneni funzionali

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents warrants proper technique, thorough training, and proper personal protective equipment. To aid in the training of researchers using organolithium reagents, a thorough, step-by-step protocol for the safe and effective use of tert-butyllithium on an inert gas line or within a glovebox is described. As a model reaction, preparation of lithium tert-butyl amide by the reaction of tert-butyl amine with one equivalent of tert-butyl lithium is presented.

The safe and proper use of organolithium reagents is described.

Un protocollo per reazioni sicuro litiazione Utilizzando organolitio Reagenti

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Un protocollo ottimizzato per la radiomarcatura efficiente di oro nanoparticelle utilizzando un<sup&gt; 125</sup&gt; Azide gruppo I-etichettati protesico

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

Il protocollo MPLEx Multi-omic nell'analisi di campioni di terreno

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological targets and processes. However, the increasing number of different tracers creates challenges for their production at radiopharmacies. While historically it has been practical to dedicate a custom-configured radiosynthesizer and hot cell for the repeated production of each individual tracer, it is becoming necessary to change this workflow. Recent commercial radiosynthesizers based on disposable cassettes/kits for each tracer simplify the production of multiple tracers with one set of equipment by eliminating the need for custom tracer-specific modifications. Furthermore, some of these radiosynthesizers enable the operator to develop and optimize their own synthesis protocols in addition to purchasing commercially-available kits. In this protocol, we describe the general procedure for how the manual synthesis of a new PET tracer can be automated on one of these radiosynthesizers and validated for the production of clinical-grade tracers. As an example, we use the ELIXYS radiosynthesizer, a flexible cassette-based radiochemistry tool that can support both PET tracer development efforts, as well as routine clinical probe manufacturing on the same system, to produce [18F]Clofarabine ([18F]CFA), a PET tracer to measure in vivo deoxycytidine kinase (dCK) enzyme activity. Translating a manual synthesis involves breaking down the synthetic protocol into basic radiochemistry processes that are then translated into intuitive chemistry &#34;unit operations&#34; supported by the synthesizer software. These operations can then rapidly be converted into an automated synthesis program by assembling them using the drag-and-drop interface. After basic testing, the synthesis and purification procedure may require optimization to achieve the desired yield and purity. Once the desired performance is achieved, a validation of the synthesis is carried out to determine its suitability for the production of the radiotracer for clinical use.

Automation of a Positron-emission Tomography PET Radiotracer Synthesis Protocol for Clinical Production

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

Automazione di un positrone-emissione di positroni (PET) radiotraccitore sintesi protocollo per la produzione di clinica

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

Terapia genica ispirato Polycation rivestimento per la protezione di nanostrutture di DNA Origami

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...