S.E. riconosce il sostegno dal fondo di Duke dotazione speciale iniziativa, fondo di Wells Fargo, PhRMA Foundation, nonché avvio fondi presso la University of North Carolina, Charlotte.

1. sintesi del complesso BSA-Au(III)
<ol><li>Sciogliere 25 mg di BSA in 1 mL di acqua di grado di cromatografia liquida ad alte prestazioni (HPLC) in un flaconcino di reazione da 5 mL. Nota: La soluzione dovrebbe apparire chiara.</li>
	<li>Sciogliere triidrato di oro (III) cloruro (acido cloroaurico) ad una concentrazione di 5 millimetri di acqua di grado HPLC. Nota: La soluzione appare gialla. Soluzione di acido cloroaurico preparata a questa concentrazione si tradurrà in una BSA al rapporto tra Au 01:13.<ol>
<li>In alternativa, preparare una soluzione di acido cloroaurico con una concentrazione di ovunque tra 0,38 mM (BSA:Au = 1:1) fino a 20 mM (BSA:Au = 01:50) in HPLC del grado dell'acqua. Nota: Diversi rapporti di BSA all'oro si tradurrà in modelli drasticamente diversa fluorescenza rossa della mappa eccitazione-emissione.</li>
	</ol>
</li>
	<li>Collocare il flaconcino di reazione di BSA in bagnomaria a 37 ° C e mescolare vigorosamente a 750 giri/min con un agitatore magnetico.</li>
	<li>Subito dopo inizia l'agitazione, aggiungere 1 mL di acido cloroaurico alla soluzione. Il colore della soluzione dovrebbe trasformare da incolore a giallo.</li>
	<li>Mescolare il composto per 2 min a 37 ° C e a 750 giri/min con un agitatore magnetico.</li>
	<li>Nella fiala di reazione, aggiungere 100 μL di 1 M NaOH alla soluzione per portare il pH a 12. Nota: Immediatamente dopo l'aggiunta di NaOH, la soluzione dovrebbe scurire leggermente al giallo-marrone e poi tornare indietro al giallo.</li>
	<li>Continuare a mescolare a 750 giri/min per 2 h e a 37 ° C. La soluzione dovrebbe cambiare lentamente dal giallo ad un colore giallo/marrone scuro. Questo cambiamento di colore indica la formazione del rosso fluorescenti BSA-Au(III) complesso.</li>
	<li>Lasciare riposare a temperatura ambiente per 2 giorni, il campione e la soluzione continuerà a scurire ad un marrone ambrato e aumenterà l'intensità di fluorescenza.<ol>
<li>In alternativa, lasciare il campione continuare a mescolare a 37 ° C per 12 h più come il colore della soluzione si evolve ad un'ambra marrone.</li>
	</ol>
</li>
</ol>2. sintesi di BSA-Cu(II)-Au(III)
<ol><li>Sciogliere 25 mg di BSA in 1 mL di acqua di grado HPLC. La soluzione dovrebbe apparire chiara.</li>
	<li>Sciogliere il rame (II) cloruro diidrato in acqua di grado HPLC a una concentrazione di 5 mM. La soluzione dovrebbe apparire la luce blu.</li>
	<li>Aggiungere 1 mL di soluzione acquosa di BSA per un flaconcino di reazione da 5 mL e posto in un bagno di acqua a 37 ° C. Mescolare il composto a 750 giri/min.</li>
	<li>Immediatamente aggiungere 0,5 mL della soluzione di rame (II) cloruro diidrato nel flaconcino di reazione e mescolare per 2 min. La soluzione rimarrà luce blu.</li>
	<li>Aggiungere 75 µ l di 1 M NaOH per portare il pH a 12 e lasciar per mescolare per 2 h. La soluzione diventerà viola.</li>
	<li>Sciogliere l'acido cloroaurico in acqua di grado HPLC a una concentrazione di 5 mM.</li>
	<li>Aggiungere 0,5 mL di acido cloroaurico acquosa nel flaconcino di reazione e regolare il pH torna a 12 utilizzando 1 M NaOH.</li>
	<li>Mescolare la miscela di reazione per 2 h. Nota: La soluzione dovrebbe evolvere in un color marrone.</li>
</ol>3. sintesi di BSA-Ni(II)-Au(III)
<ol><li>Sciogliere 25 mg di BSA in 1 mL di acqua di grado HPLC. La soluzione dovrebbe apparire chiara.</li>
	<li>Sciogliere il nichel (II) cloruro esaidrato in acqua di grado HPLC a una concentrazione di 5 mM. La soluzione dovrebbe apparire la luce verde.</li>
	<li>Aggiungere 1 mL di soluzione acquosa di BSA per un flaconcino di reazione da 5 mL e porre in un bagno di acqua a 37 oC. mescolare la miscela a 750 giri/min.</li>
	<li>Immediatamente aggiungere 0,5 mL della soluzione di nichel (II) cloruro esaidrato flaconcino di reazione e mescolare per 2 min. Nota: La soluzione rimarrà luce verde.</li>
	<li>Aggiungere 75 µ l di 1 M NaOH per portare il pH a 12 e lasciar per mescolare per 2 h. Nota: La soluzione diventa giallo scuro.</li>
	<li>Sciogliere l'acido cloroaurico in acqua di grado HPLC a una concentrazione di 5 mM.</li>
	<li>Aggiungere 0,5 mL di acido cloroaurico acquosa nel flaconcino di reazione e regolare il pH torna a 12.</li>
	<li>Mescolare la miscela di reazione per 2 h. Nota: La soluzione dovrebbe evolvere in un color marrone.</li>
</ol>4. sintesi di [Cys34-capped-BSA]-Au(III)
<ol><li>Sciogliere 2 mg di N-ethylmaleimide (NEM) in 1 mL di tampone fosfato salino (PBS, pH 7.4).</li>
	<li>Sciogliere 2 mg di BSA in 1 mL di soluzione di PBS-NEM.</li>
	<li>Trasferire la soluzione in un flaconcino di reazione da 5 mL e mescolare a 20 ° C a 500 giri/min per 1 h.</li>
	<li>Dializzare la soluzione utilizzando 12 tubi di dialisi di kDa in 500 mL di PBS, agitazione a 50 giri/min con un agitatore magnetico durante la notte per rimuovere NEM non reagito.</li>
	<li>Sciogliere l'acido cloroaurico in PBS per una concentrazione di 0,4 mM. Nota: La soluzione sarà un giallo tenue.</li>
	<li>Trasferire il flaconcino di reazione a un bagno di acqua a 37 ° C. Mescolare a 750 giri/min.</li>
	<li>Immediatamente aggiungere 1 mL di soluzione di acido cloroaurico nel flaconcino di reazione e lasciar per mescolare per 2 min.</li>
	<li>Aggiungere 75 μL di 1 M NaOH al flaconcino di reazione per portare il pH a 12 e lasciar per mescolare per 2 h.</li>
</ol>5. sintesi di [all-thiol-capped-BSA]-Au(III)
<ol><li>Preparare una soluzione 2 M urea e 50mm di bicarbonato di ammonio (NH4HCO3, pH 8.0) in acqua di grado HPLC.</li>
	<li>Sciogliere 3,3 mg di BSA in 1 mL della soluzione precedente e trasferimento di un flaconcino di reazione da 5 mL.</li>
	<li>Fare una soluzione stock di 0,25 M tris(2-carboxyethyl) fosfina (TCEP) sciogliendo 62,5 mg di TCEP in 1 mL di acqua per HPLC.</li>
	<li>Aggiungere la soluzione madre di TCEP nel flaconcino di reazione fino a quando la concentrazione finale di TCEP è di 8 mM.</li>
	<li>Incubare la soluzione in un bagno di acqua per 1 h a 50 ° C. Mescolare a 500 giri/min con un agitatore magnetico.</li>
	<li>Lasciare la soluzione completamente raffreddare a temperatura ambiente.</li>
	<li>Preparare una soluzione stock di 100 mM NEM sciogliendo 12,5 mg di NEM in 1 mL di acqua di grado HPLC.</li>
	<li>Aggiungere la soluzione madre di NEM nel flaconcino di reazione fino a quando la concentrazione finale di NEM è di 16 mM.</li>
	<li>Lasciare agire la soluzione combinare per 2 h a 20 ° C. Mescolare a 500 giri/min.</li>
	<li>Dializzare la soluzione utilizzando un tubo di dialisi di 12 kDa, mescolando la soluzione a 50 giri/min con un agitatore magnetico pernottamento in 500 mL di 50mm NH4HCO3 per rimuovere l'eccesso TCEP, NEM e urea.</li>
	<li>Spostare il flaconcino di reazione in un bagno di acqua a 37 ° C e mescolare a 750 giri/min.</li>
	<li>Sciogliere l'acido cloroaurico in acqua di grado HPLC a una concentrazione di 0,66 mM.</li>
	<li>Immediatamente aggiungere 1 mL di soluzione di acido cloroaurico nel flaconcino di reazione e lasciar per mescolare per 2 min.</li>
	<li>Aggiungere 1 M NaOH fino a quando il pH della soluzione è 12 e lasciare che la soluzione di continuare a mescolare per 2 h.</li>
</ol>

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I composti di BSA-Au(III) preparati a pH 12 presentano fluorescenza rossa ad una lunghezza d'onda di emissione di λem= 640 nm quando eccitato con raggi ultravioletti (UV) luce λex= 365 nm (Figura 1A). L'emergere di fluorescenza rossa è un processo lento e avrà un paio di giorni a temperatura ambiente per aumentare a un'intensità massima. In esecuzione della reazione a 37 ° C produrrà i risultati ottimali, anche se alta temperatura può essere utilizzato ...

Un composto di BSA-Au esibendo un ultravioletto (UV)-eccitabile fluorescenza rossa, con notevole spostamento di stokes, è stato originariamente sintetizzato da Xie et al. 1. la fluorescenza rossa univoco e stabile può trovare varie applicazioni in campi quali rilevamento2,3,4,5,6,7o imaging nanomedicina8 ,9,10,11,12,13. Questo composto è stato studiato estesamente da molti ricercatori nel campo delle nano-scienze in ultimi anni14,15,16. Il composto di BSA-Au è stato interpretato come Au25 nanocluster. L'obiettivo del metodo presentato è per esaminare questo composto in dettaglio e per capire l'origine della fluorescenza rossa. Seguendo l'approccio presentato, la presenza di più siti di legame di Au e l'origine della fluorescenza, alternativa per la nucleazione di singolo sito di nanocluster di Au25 , possono essere illustrate. Lo stesso approccio può essere impiegato per studiare come altre proteine17,18,19 complessato con Au(III) può cambiare le loro proprietà intrinseche, fluorescente.
La sintesi del composto rosso fluorescente BSA-Au richiede un controllo stretto dei rapporti molari di BSA per Au (BSA:Au) per massimizzare l'intensità della fluorescenza e la posizione dei picchi a eccitazione-emissione mappa (EEM)20. Si può dimostrare che più siti di legame esistano per Au(III) per l'associazione, tra cui il frammento di asparagina (o frammento di Asp, i primi quattro residui dell'amminoacido al N-terminale di BSA)21,22. Dell'aminoacidoth 34 di BSA (Cys-34) è inoltre indicato per coordinare Au(III) e di essere coinvolti nel meccanismo del rosso fluorescence([Cys34-capped-BSA]-Au(III))20. Su tutti i ponti disolfuro Cys Cys di sfaldatura e tappatura tutti fluorescenza di tioli, rosso non è prodotta ([all-thiol-capped-BSA]-Au(III)). Questo indica la necessità di legami disolfuro Cys Cys come il sito di legame di Au(III) per produrre la fluorescenza rossa.
Tecniche di chimica della proteina non sono stati ampiamente utilizzati per studiare i complessi di BSA-Au(III) della comunità di nano-scienza. Tuttavia, sarebbe prezioso per impiegare queste tecniche per capire alcuni aspetti di questi complessi, nonché di acquisire una comprensione dettagliata delle sedi del legame di Au(III) in BSA. Questo articolo è destinato a mostrare alcune di queste tecniche.

<table>
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA), 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A5611</td>
			<td></td>
		</tr>
		<tr>
			<td>gold (III) chloride trihydrate, 99.9%</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper (II) chloride dihydrate, 99.999%</td>
			<td>Sigma-Aldrich</td>
			<td>459097</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel (II) chloride hexahydrate, 99.9%</td>
			<td>Sigma-Aldrich</td>
			<td>654507</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Ethylmaleimide (NEM), &#62;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>4259</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &#62;98.0%</td>
			<td>Sigma-Aldrich</td>
			<td>C4706</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide, &#62;98.0%</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea, 99.5%</td>
			<td>Chem-Implex Int&#39;l</td>
			<td>30142</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffered saline (PBS)</td>
			<td>Corning</td>
			<td>MT21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate, 99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
	</tbody>
</table>

luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)

Lo scopo dei protocolli presentati è quello di studiare il processo di associazione Au(III) a BSA, cedendo la fluorescenza rossa indotta da cambiamento di conformazione (λem = 640 nm) dei complessi BSA-Au(III). Il metodo consente di regolare il pH per mostrare che l'emersione della fluorescenza rossa è correlato con le transizioni di equilibrio pH-indotta delle conformazioni BSA. Rosso fluorescente BSA-Au(III) complessi possono essere formati solo con un adeguamento del pH pari o superiore a 9.7, che corrisponde alla conformazione "A-form" di BSA. Il protocollo per regolare la BSA al rapporto molare Au e per monitorare il corso di tempo del processo di associazione Au(III) è descritto. Il numero minimo di Au(III) a BSA, per produrre la fluorescenza rossa, è meno di sette. Descriviamo il protocollo nei passaggi per illustrare la presenza di più siti di legame di Au(III) in BSA. Prima, con l'aggiunta di rame (Cu(II)) o nichel (cationi Ni(II)) seguiti da Au(III), questo metodo si rivela un sito di legame per Au(III) che non è il fluoroforo rosso. In secondo luogo, modificando tiolo tappatura agenti BSA, viene rivelato un altro sito di legame di Au(III) nonfluorophore-formanti. In terzo luogo, i dominî di BSA di sfaldatura e tappatura dei legami disolfuro, sono illustrati i possibili siti di associazione Au(III). Il protocollo descritto, per controllare le conformazioni di BSA e l'associazione Au(III), può essere generalmente applicato per studiare le interazioni di altre proteine e cationi metallici.

Dalla fluorescenza del complesso BSA-Au(III), è stato osservato che la conversione della fluorescenza intrinseca blu di BSA (λem = 400 nm) a fluorescenza rossa (λem = 640 nm) si verifica a circa pH 9.7 attraverso un equilibrio transizione (Figura 1). EEM di BSA-Au(III) alle diversa BSA a rapporti molari Au è illustrato nella Figura 2, e questi dati mostrano come alterare i rapporti mola...

Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

I protocolli per studiare l'associazione di oro cationi (Au(III)) a varie conformazioni di albumina di siero bovino (BSA) come pure per caratterizzare la fluorescenza di BSA-Au unica dipendente conformazionale sono presentati.

Luminophore formazione in varie conformazioni di albumina di siero bovino di legame dell'oro

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

luminophore-formation-various-conformations-bovine-serum-albumin

Research

JoVE Journal

Chemistry

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

6.8K Views.  University of North Carolina. I protocolli per studiare l'associazione di oro cationi (Au(III)) a varie conformazioni di albumina di siero bovino (BSA) come pure per caratterizzare la fluorescenza di BSA-Au unica dipendente conformazionale sono presentati.

Video: Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

LabVIEW a comando nanolitri osmometro Novel per le indagini Protein Ice rilegatura

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

Ricerca

Chimica

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Su larga scala e non mirato Profiling Metabolomica di siero da Ultra Performance Liquid Chromatography-Spettrometria di Massa (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Legame covalente di BMP-2 su superfici utilizzando un auto-assemblato Monolayer Approccio

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

Forno a microonde assistita Funzionalizzazione di poli (etilene glicole) e On-resina Peptidi per l&#39;uso a polimerizzazione a catena ed idrogel Formazione

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

Formazione di strutture biomolecolari ordinate dalla Self-assemblaggio di piccoli peptidi

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Determinazione dei piani di legame del ghiaccio delle proteine antigelo mediante affinità del piano di ghiaccio basata sulla fluorescenza

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Acqua in emulsioni olio: un nuovo sistema di montaggio idrosolubili proteine ​​clorofilla vincolante con pigmenti idrofobi

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Radiomarcatura e quantificazione dei livelli cellulari di fosfoinositidi da alte prestazioni cromatografia liquida accoppiata flusso scintillazione

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

In Situ Caratterizzazione di idrate proteine ​​in acqua da Salvi e TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

DNA-magnetoscopico associazione analisi di dispersione della luce dinamica ed elettroforetica

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...