S.E. reconhece o apoio do fundo especial iniciativa Duke, Wells Fargo fundo, PhRMA Foundation, bem como fundos de inicialização da Universidade da Carolina do Norte, Charlotte.

1. síntese do complexo de BSA-Au(III)
<ol><li>Dissolva 25 mg de BSA em 1 mL de água de grau de cromatografia líquida de alto desempenho (HPLC) em um frasco de reação de 5 mL. Nota: A solução deve aparecer claramente.</li>
	<li>Dissolva o ouro (III) cloreto trihidrato (ácido chloroauric) a uma concentração de 5 mM em água de grau HPLC. Nota: A solução deve aparecer amarela. Solução de Ácido cloroáurico preparada nessa concentração resultará em uma BSA, à relação entre Au 01:13.<ol>
<li>Em alternativa, preparar uma solução de Ácido cloroáurico com uma concentração de em qualquer lugar entre 0,38 mM (BSA:Au = 1:1) de 20 mM (BSA:Au = 01:50) em HPLC grau água. Nota: Diferentes rácios de BSA de ouro irão resultar em padrões de fluorescência vermelha drasticamente diferente do mapa de excitação-emissão.</li>
	</ol>
</li>
	<li>Coloque o frasco de reação da BSA em banho maria a 37 ° C e agitar vigorosamente a 750 rpm utilizando um agitador magnético.</li>
	<li>Imediatamente após a agitação começa, adicione 1 mL de Ácido cloroáurico à solução. A cor da solução deve se transformar de clear para amarelo.</li>
	<li>Agite a mistura por 2 min a 37 ° C e a 750 rpm utilizando um agitador magnético.</li>
	<li>Dentro do frasco de reação, adicione 100 μL de 1 M de NaOH à solução para trazer o pH para 12. Nota: Imediatamente depois de NaOH é adicionado, a solução deve escurecer ligeiramente para um amarelo-marrom e então entregue-se ao amarelo.</li>
	<li>Continuar a agitar a 750 rpm por 2 h e a 37 ° C. A solução deve lentamente mudar de amarelo para cor amarelo/marrom escura. Esta mudança de cor indica a formação da BSA-Au(III) fluorescentes vermelhas complexas.</li>
	<li>Permitir que a amostra sentar-se à temperatura ambiente por 2 dias e a solução continuará a escurecer para um marrom âmbar e irá aumentar a intensidade de fluorescência.<ol>
<li>Como alternativa, deixe a amostra continuar a agitar a 37 ° C por 12 h mais como a cor da solução evolui para um âmbar marrom.</li>
	</ol>
</li>
</ol>2. síntese de BSA-Cu(II)-Au(III)
<ol><li>Dissolva 25 mg de BSA em 1 mL de água de grau HPLC. A solução deve aparecer claramente.</li>
	<li>Dissolva o cobre (II) cloreto hidratado em água de grau HPLC para uma concentração de 5 mM. A solução deve aparecer a luz azul.</li>
	<li>Adicione 1 mL da solução aquosa BSA para um frasco de reação de 5 mL e colocar em banho maria a 37 ° C. Agite a mistura em 750 rpm.</li>
	<li>Imediatamente Adicione 0,5 mL da solução de-hidratado cobre (II) cloreto ao frasco de reação e misture por 2 min. A solução permanecerá luz azul.</li>
	<li>Adicione 75 μL de 1 M NaOH para trazer o pH para 12 e permitem misturar por 2 h. A solução se tornará roxa.</li>
	<li>Dissolva o Ácido cloroáurico em água de grau HPLC para uma concentração de 5 mM.</li>
	<li>Adicionar 0,5 mL de Ácido cloroáurico aquosa ao frasco de reação e ajustar o pH volta a 12 usando 1 M de NaOH.</li>
	<li>Agite a mistura de reação para h 2. Nota: A solução deve evoluir para uma cor marrom.</li>
</ol>3. síntese de BSA-Ni(II)-Au(III)
<ol><li>Dissolva 25 mg de BSA em 1 mL de água de grau HPLC. A solução deve aparecer claramente.</li>
	<li>Dissolva o cloreto de níquel (II) hexaidratado em água de grau HPLC para uma concentração de 5 mM. A solução deve aparecer a luz verde.</li>
	<li>Adicione 1 mL da solução aquosa BSA para um frasco de reação de 5 mL e coloque num banho de água em 37 oC. agitar a mistura em 750 rpm.</li>
	<li>Imediatamente Adicione 0,5 mL da solução de hexaidrato cloreto de níquel (II) para o frasco de reação e misture por 2 min. Nota: A solução permanecerá luz verde.</li>
	<li>Adicione 75 μL de 1 M NaOH para trazer o pH para 12 e permitem misturar por 2 h. Nota: A solução se tornará amarelo escuro.</li>
	<li>Dissolva o Ácido cloroáurico em água de grau HPLC para uma concentração de 5 mM.</li>
	<li>Adicionar 0,5 mL de Ácido cloroáurico aquosa ao frasco de reação e ajustar o pH volta a 12.</li>
	<li>Agite a mistura de reação para h 2. Nota: A solução deve evoluir para uma cor marrom.</li>
</ol>4. síntese de [Cys34-capped-BSA]-Au(III)
<ol><li>Dissolva 2 mg de N-proteÃ (NEM) em 1 mL de salina tamponada fosfato (PBS, pH 7,4).</li>
	<li>Dissolva 2 mg de BSA em 1 mL de solução de PBS-NEM.</li>
	<li>Transferir a solução para um frasco de reação de 5 mL e agitar a 20 ° C a 500 rpm por 1h.</li>
	<li>Dialize a solução usando 12 kDa tubulação da diálise em 500 mL de PBS, agitando a 50 rpm com um agitador magnético durante a noite para remover NEM não tenha reagido.</li>
	<li>Dissolva o Ácido cloroáurico em PBS a uma concentração de 0,4 mM. Nota: A solução será um amarelo fraco.</li>
	<li>Transferência do frasco de reação para um banho de água a 37 ° C. Misture a 750 rpm.</li>
	<li>Imediatamente adicione 1 mL da solução de Ácido cloroáurico ao frasco de reação e permitir a mistura por 2 min.</li>
	<li>Adicione 75 μL de 1 M de NaOH no frasco de reação para trazer o pH para 12 e permitem misturar por 2 h.</li>
</ol>5. síntese de [all-thiol-capped-BSA]-Au(III)
<ol><li>Prepare uma solução de 2 M 50 milímetros e ureia bicarbonato de amónio (NH4HCO3, pH 8.0) em água de grau HPLC.</li>
	<li>Dissolva a 3,3 mg de BSA em 1 mL da solução acima e transferir para um frasco de reação de 5 mL.</li>
	<li>Fazer uma solução stock de 0,25 M tris(2-carboxyethyl) fosfina (TCEP) pela dissolução de 62,5 mg de TCEP em 1 mL de água HPLC.</li>
	<li>Adicione a solução-mãe de TCEP ao frasco de reação até que a concentração final de TCEP é de 8 mM.</li>
	<li>Incubar a solução em um banho de água durante 1 h a 50 ° C. Misture a 500 rpm utilizando um agitador magnético.</li>
	<li>Deixe a solução arrefecer completamente à temperatura ambiente.</li>
	<li>Prepare uma solução stock de 100mm NEM pela dissolução de 12,5 mg de NEM em 1 mL de água de grau HPLC.</li>
	<li>Adicione a solução stock de NEM ao frasco de reação até que a concentração final de NEM é 16 milímetros.</li>
	<li>Permitir que a solução combinar para 2 h a 20 ° C. Misture a 500 rpm.</li>
	<li>Dialize a solução usando um tubo de diálise 12 kDa, agitando a solução a 50 rpm com um agitador magnético durante a noite em 500 mL de 50mm NH4HCO3 para remover o excesso TCEP, NEM e ureia.</li>
	<li>Mover o frasco de reação para um banho-maria a 37 ° C e agitar a 750 rpm.</li>
	<li>Dissolva o Ácido cloroáurico em água de grau HPLC para uma concentração de 0,66 mM.</li>
	<li>Imediatamente adicione 1 mL da solução de Ácido cloroáurico ao frasco de reação e permitir a mistura por 2 min.</li>
	<li>Adicionar 1 M de NaOH até que o pH da solução é 12 e permitir que a solução continuar a misturar por 2 h.</li>
</ol>

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Os autores não têm nada para divulgar.

Os compostos de BSA-Au(III) preparados em pH 12 apresentam fluorescência vermelha em um comprimento de onda de emissão de λem= 640 nm quando excitadas com ultravioleta (UV) luz λex= 365 nm (figura 1A). O aparecimento de fluorescência vermelha é um processo lento e levará alguns dias à temperatura ambiente para aumentar a uma intensidade máxima. Executando a reação a 37 ° C irá produzir os melhores resultados, apesar de temperatura mais elevada pode...

Um composto de BSA-Au exibindo uma radiação ultravioleta (UV)-fluorescência vermelha excitável, com notável stokes turno, tem sido sintetizado originalmente por Xie et al. 1. a fluorescência vermelha única e estável pode encontrar várias aplicações em campos como a detecção de3,2,4, imagem5,6,7ou nanomedicina8 ,9,10,11,12,13. Este composto tem sido estudado extensivamente por muitos pesquisadores no campo da nano-ciência em anos recentes14,15,16. O composto de BSA-Au tem sido interpretado como Au25 nanoclusters. O objetivo do método apresentado é para examinar este composto em detalhe e para compreender a origem da fluorescência vermelha. Seguindo a abordagem apresentada, a presença de vários sites de ligação do Au e a origem da fluorescência, alternativa para a nucleação de single-site de Au25 nanoclusters, podem ser ilustrados. A mesma abordagem pode ser utilizada para estudar como outras proteínas17,18,19 complexado com Au(III) pode alterar suas propriedades intrínsecas, fluorescentes.
A síntese do composto fluorescente vermelha BSA-Au requer um controle estreito dos rácios molares da BSA para Au (BSA:Au) para maximizar a intensidade da fluorescência e a localização dos picos a excitação-emissão de mapa (EEM)20. Pode-se demonstrar que vários sites de vinculação existem para Au(III) vincular, incluindo o fragmento de asparagina (ou fragmento de Asp, os primeiro de quatro resíduos de aminoácidos no N-terminal da BSA)21,22. O aminoácido 34th de BSA (Cys-34) também é mostrado para coordenar Au(III) e de estar envolvido no mecanismo do fluorescence([Cys34-capped-BSA]-Au(III)) vermelho20. Fendendo a todas as ligações de bissulfeto Cys-Cys e tampando todos fluorescência de tióis, vermelho não é produzido ([all-thiol-capped-BSA]-Au(III)). Isto indica a necessidade de ligações de bissulfeto Cys-Cys como o sítio de ligação do Au(III) para produzir a fluorescência vermelha.
Técnicas de química de proteínas não foram amplamente utilizadas para estudar os complexos de BSA-Au(III) na Comunidade de nano-ciência. No entanto, é importante empregar estas técnicas para entender alguns aspectos destes complexos, bem como para obter uma compreensão detalhada dos locais de ligação Au(III) da BSA. Este artigo pretende mostrar algumas dessas técnicas.

<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), &gt;99.0%</td><td>Sigma-Aldrich</td><td>4259</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &gt;98.0%</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Sodium hydroxide, &gt;98.0%</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Urea, 99.5%</td><td>Chem-Implex Int&#039;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)

O objetivo dos protocolos apresentados é estudar o processo de ligação de Au(III) a BSA, rendendo fluorescência vermelha induzida pela mudança de conformação (λem = 640 nm) de complexos de BSA-Au(III). O método ajusta o pH para mostrar que o surgimento da fluorescência vermelha está correlacionado com as transições de equilíbrio de pH-induzida de conformações da BSA. Fluorescente vermelha BSA-Au(III) complexos só podem ser formados com um ajuste de pH igual ou superior a 9,7, que corresponde a conformação de "Uma-forma" da BSA. O protocolo para ajustar a BSA a razão molar de Au e para monitorar o tempo-curso do processo de vinculação de Au(III) é descrito. O número mínimo de Au(III) por BSA, para produzir a fluorescência vermelha, tem menos de sete anos. Descrevemos o protocolo em etapas para ilustrar a presença de vários sites de ligação Au(III) em BSA. Primeiro, pela adição de cobre (Cu(II)) ou níquel (Ni(II)) cações seguidas por Au(III), este método revela um sítio de ligação para Au(III) que não é o fluoróforo vermelho. Em segundo lugar, modificando BSA tiol tampando agentes, outro sítio de ligação de Au(III) nonfluorophore-formando é revelado. Em terceiro lugar, alterando a conformação de BSA por clivagem e nivelamento das ligações de bissulfeto, Au(III) possível ligação site é ilustrado. O protocolo descrito, para controlar as conformações de BSA e Au(III) ligação, pode ser aplicado geralmente para estudar as interações de outras proteínas e cátions metálicos.

De fluorescência do complexo BSA-Au(III), tem sido observado que a conversão da fluorescência azul intrínseca de BSA (λem = 400 nm) para fluorescência vermelha (λem = 640 nm) ocorre no sobre pH 9,7 através de um equilíbrio transição (Figura 1). EEM de BSA-Au(III) no BSA diferente rácios molar Au é mostrado na Figura 2, e esses dados mostram como alterar as proporções molares ...

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

Os protocolos para estudar a ligação de ouro cátions (Au(III)) para várias conformações de albumina de soro bovino (BSA), bem como para caracterizar a fluorescência de BSA-Au única dependente conformacional são apresentadas.

Lumíforo formação em várias conformações de albumina de soro bovino por ligação de Gold(III)

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 ...

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Research

JoVE Journal

Chemistry

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

6.8K visualizações.  University of North Carolina. Os protocolos para estudar a ligação de ouro cátions (Au(III)) para várias conformações de albumina de soro bovino (BSA), bem como para caracterizar a fluorescência de BSA-Au única dependente conformacional são apresentadas.

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

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

Iridium (III) luminescentes Sonda para detecção da proteína maláricos Biomarker proteína rica em histidina-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Química

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

Au-Interação de Slp1 Polímeros e monocamada de<em&gt; Lysinibacillus sphaericus</em&gt; JG-B53 - QCM-D, o ICP-MS e AFM como Ferramentas para Estudos biomolécula metal-

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Quantificação de Proteome-largo de rotulagem homogeneidade a nível da molécula

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Bioquímica

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Uma rápida e quantitativa método fluorimétrico para-Targeting proteína pequena molécula Rastreio de drogas

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Bioengenharia

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

<meta charset="utf8"></head><body>Determinação das concentrações de proteína total e proteína bioativa no colostro bovino

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Determinação das concentrações de proteína total e proteína bioativa no colostro bovino

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a ...

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable red luminescence (&#955;em = 640 nm), with unusual Stokes shifts compared to the innate UV/blue fluorescence arising from the aromatic residues. Red-luminescent complexes are formed in highly alkaline conditions above pH 10 and require a conformation change within the protein to occur. In addition, preservation of Cys-Cys disulfide bonds in BSA is necessary to obtain this red luminescence. In order to understand the mechanism of this luminescence, elucidation of the luminophore-forming Au(III) binding site is essential. A facile way to assess the luminophore-forming site would be to (1) predictably fragment the protein by enzymatic digestion, (2) react the obtained fragments with Au(III), then (3) perform gel electrophoresis to observe the well-separated fragment bands and analyze the in-gel red luminescence. However, due to the alkaline conditions and the reaction with metal cations, new limited proteolysis techniques and gel electrophoresis conditions must be applied. Particularly, the presence of metal cations in gel electrophoresis can make the band separations technically difficult. We describe this new protocol in steps to identify the red-luminophore-forming metal binding domain in BSA. This protocol can thus be applied for analyzing protein fragments that must remain in a non-denatured or a partially denatured state, in the presence of metal cations. Because the majority of proteins need metal cations to function, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. This method, on the other hand, could be used in supplement to study the interactions of proteins with metal cations without requiring the protein crystallization and at a desired pH condition.

<meta charset="utf8"></head><body>Proteólise limitada e eletroforese em gel na presença de cátions metálicos: domínio luminescente de ligação a AuIII em albuminas séricas

We present a protocol for studying the binding domain of Au(III) in bovine serum albumin (BSA).

Proteólise Limitada e Eletroforese em Gel na Presença de Cátions Metálicos: Domínio Luminescente de Ligação a Au(III) em Albuminas Séricas

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Síntese de nanoaglomerados de ouro emissor próximos para aplicações biológicas

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

Caracterização da automontagem reversível dependente de pH de colóides de ouro revestidos com beta-amiloide 1-40

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Lumíforo formação em várias conformações de albumina de soro bovino por ligação de Gold(III)

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