Esta pesquisa foi apoiada pela ISF, NSF, e ERC. Nós gostaríamos de agradecer a ajuda técnica com a fase de temperatura de Randy Milford, Michael Koren, Shafer Doug, e Dennison Jeremy. Assistência ao desenvolvimento de software foi fornecido pela Ou Chen, Xu Di, Sannareddy Rajesh, e Bhattachary Sumit. Gostaríamos de agradecer aos nossos colaboradores o professor Peter L. Davies e Dr. Laurie A. Graham para a proteína IBP MP e discussões úteis. Agradecemos também laboratório membros Dr. Maya Bar-Dolev, Yangzhong Qin, Dr. Yeliz Celik, Dr. Natalya Pertaya, Ortal Mizrahy, e Guy Shlomit para seu feedback do usuário.

0. Procedimentos preliminares <ol><li> Capilar de vidro para injectáveis ​​solução. Usando um extrator capilar (Narishige, Tóquio, Japão), preparar uma pipeta afiada com uma abertura fina a partir de um tubo capilar de vidro micro (Marca GMBH, Wertheim, Alemanha). O tamanho da abertura deve ser verificada por passagem de ar através do capilar para obter borbulhante fina em água limpa. Se o capilar é fechado, então pode-se abri-lo por quebrar sua borda. Isto pode ser feito por prensagem ou arranhar o suavemente contra a água contendo paredes do tubo. Prepare o capilar tal que a abertura está quase bloqueada, mas é suficientemente aberta para permitir a formação de sub-milímetros bolhas. </li><li> Limpeza de cobre disco. Sonicar os discos de cobre durante 10 min em 0,1% de Micro-90 sabão (Cole-Parmer, Vernon Hills, Illinois, EUA), depois lava-se com água duplamente destilada. Introduzir os discos em uma solução de isopropanol (técnico) e sonicar novamente durante 10 min. Barbatanaaliado, seque os discos usando ar filtrado. Esta fase de limpeza é crítico para evitar a contaminação entre IBP experimentos. </li><li> De camada dupla montagem lamela. A montagem lamela foi preparada para permitir a observação da amostra sem condensação de humidade sobre a superfície do vidro de cobertura. Isto foi conseguido através da colocação de uma partícula de Drierite (WA Hammond Drierite, Xenia, Ohio, EUA), (2 mm de diâmetro) entre duas lamelas, que foram, em seguida, colados com uma pistola de cola quente. Esta configuração impedido condensação que poderia bloquear a vista quando a amostra foi arrefecida a baixa temperatura e elimina a necessidade de soprar ar seco para a janela de observação. </li></ol> 1. Estágio de resfriamento Set-up <ol><li> Ligue a entrada do fluxo de água e de saída da fase de arrefecimento a 4 mm de diâmetro interno Tygon tubos (Saint-Gobain, Paris, França), e ligar o tubo de entrada de água do fluxo de uma bomba de água. </li><li> Ligar um tubo Tygon 4 milímetros de diâmetro interno para a entrada da etapa de arrefecimento tó fornecer ar seco. O ar foi seco utilizando uma coluna de linha Drierite. </li><li> Operar as bombas de ar e água. Note-se que os elementos de resfriamento não deve ser executado sem um dissipador de calor. </li><li> Ligue o controlador de temperatura, câmera e rotineira LabVIEW. </li></ol> 2. Preparação da Amostra <ol><li> Colocar uma gota 3-4 ul de imersão em óleo B (laboratórios Cargille, Cedar Grove, New Jersey, EUA), no lado de trás de um disco de 7 mm de diâmetro de cobre com 500 furos perfurados uM através do disco. </li><li> Posicionar o disco de cobre na fase de arrefecimento, com o lado voltado para baixo de imersão em óleo. </li><li> Ligar o tubo capilar (a ponta chanfrada) para um tubo de 0,7 milímetros Tygon diâmetro interno ligada na outra extremidade a uma seringa de vidro de 2 ml (Poulten-Graf, Wertheim, Alemanha). </li><li> Antes de utilizar o tubo capilar, verificar a pequena abertura do capilar de forma que a abertura é um tamanho apropriado (ver os procedimentos preliminares). </li><li> Insira lentamente o glass capilar para o tubo de amostra preparada IBP proteína (2,4 uM Mp IBP-GFP em 20 mM de CaCl 2 e 25 mM Tris-HCl a pH 8, ver referência 10 para os pormenores de preparação) e puxar a seringa de vidro até que o capilar de vidro contém 0,1 ul da solução de proteína. </li><li> Iniciar a gravação de vídeo através do software LabVIEW. </li><li> Inserir a ponta afiada do capilar de vidro (contendo a solução de proteína) em um dos furos do disco de cobre na fase de arrefecimento. </li><li> Enquanto observa através do microscópio (Olympus, Tokyo, Japan, objectiva 10x), cuidadosamente penetrar a camada de óleo de imersão com a ponta capilar de vidro, e pressionar a seringa de vidro (muito delicadamente) para entregar uma pequena quantidade (~ 10 nl) da proteína solução para criar uma gotícula uM 200. </li><li> Cobrir o furo na fase de arrefecimento, com a montagem de dupla camada lamela (ver os procedimentos preliminares). </li></ol> 3. Medição de Actividade TH <ol><li> Préss botão do arrefecimento e ajustar a temperatura a -40 ° C. </li><li> Inicialmente, a gota de solução será clara. A baixas temperaturas, tipicamente na gama de -30 ° C a -35 ° C, gota a muda de cor, indicando que a solução tenha sido congelada. Imediatamente após a amostra ter congelado, aumentar a temperatura lentamente até que o gelo começa a derreter a granel. Um aumento gradual da temperatura é necessária para evitar a ultrapassagem da temperatura que pode resultar na fusão completa da amostra. </li><li> Mudar para um objectivo de 50x e começar a derreter o gelo, ajustando a temperatura. Este ajuste é interativo, e os passos finais são normalmente realizadas utilizando medidas de temperatura de pequenas 0,002 ° C. Continuar a fundir antes de um único cristal permanece. O tamanho final do cristal deve ser cerca de 10 um. A temperatura mais alta à qual tenha cessado de fusão é determinado como sendo o ponto de fusão e é determinado com precisão na fase posterior análise vídeo. </li> &lt;li&gt; Ajustar a temperatura para alguns centésimos de um grau Celsius abaixo do ponto de fusão do cristal e começar uma rampa de temperatura com um atraso de 10 minutos. Ajuste a velocidade de rampa, como desejado. Durante este tempo, o cristal será exposto aos IBPS. </li><li> Após a conclusão do período de exposição de 10 min, a temperatura diminui automaticamente sob o controlo de rotina de LabVIEW. </li><li> Observar a forma dos cristais à medida que diminui a temperatura. Em algum momento, a súbita explosão de cristal de gelo pode ser observado. A temperatura à qual ocorre esta é conhecida como a temperatura de ruptura do cristal. </li><li> Usar a análise de vídeo para determinar o ponto de fusão preciso e a temperatura de ruptura. Primeiro, por meio de análise de vídeo, encontrar o ponto de fusão precisa. Recorde-se que a mais alta temperatura a que tenha deixado de fusão é determinado como sendo o ponto de fusão. Documentar este ponto de fusão, em um programa de planilha. Em seguida, determinar a temperatura de ruptura precisa de cristal, e documentar este valortambém. A diferença entre o ponto de fusão e o ponto de congelação, temperatura de ruptura ou de cristal, é a actividade de histerese térmica da solução de IBP. </li></ol> 4. Medição da Actividade TH tempo-dependente <ol><li> Seguir o protocolo descrito nas Secções 3,1-3,3 para preparar um único cristal de gelo. </li><li> Após a formação do cristal, ajustar o tempo de retardamento da rampa conforme desejado, e ligar a rampa. </li><li> A temperatura irá diminuir a uma taxa fixa (de acordo com os requisitos dos operadores) automaticamente uma vez que o tempo de atraso de rampa passou. </li><li> Documentar a temperatura a que ocorre a ruptura de cristal. Calcula-se o tempo de exposição (o tempo entre a formação de cristais e do impulso de cristal). </li><li> Repita a experiência para vários tempos de atraso e representar graficamente a actividade TH como uma função do tempo de exposição para avaliar a dependência do tempo da actividade TH. </li></ol>

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Não há conflitos de interesse declarados.

Este trabalho demonstra o funcionamento de um osmómetro nanolitro, controlado por computador, que permite medições precisas da actividade TH com controlo de temperatura excepcional. Em qualquer sistema sensível à temperatura, gradientes de temperatura indesejáveis ​​devem ser evitadas. Para evitar gradientes de temperatura no aparelho aqui apresentado, a gota de solução de teste deve ser posicionada no centro de um furo na fase de arrefecimento de cobre do disco (passo 2.7). Além disso, o cristal único deve estar no centro da gota em vez de junto das extremidades (na maioria dos casos, isto irá acontecer espontaneamente). A dependência do tempo descrito indica que a taxa de arrefecimento pode influenciar as leituras TH. Assim, sugere-se que inclua um relatório de tempo durante o qual o cristal foi exposta à solução antes do arrefecimento, assim como a velocidade de arrefecimento. Nós normalmente esperado 10 min antes da rampa para baixo a temperatura de 0.01 ° C passos cada sec 4. Os co LabVIEW controladosfase oling foi adaptado para utilização com um microscópio invertido no qual os dispositivos microfluidicos podem ser termicamente manipulado. Este sistema facilita a realização de experimentos solução de intercâmbio de cristais de gelo e IBPS marcados com eGFP 9, 10, 16. O sistema LabVIEW-controlada pode ser adaptado a uma fase Clifton, ligando o controlador de temperatura de 3040 através de um circuito de adaptação designada eléctrico. Tal sistema é operado no laboratório Davies 17. O software LabVIEW ea designada projeto de circuito elétrico adaptação para o palco Clifton estão disponíveis mediante solicitação. Em conclusão, nós descrevemos um osmómetro nanolitro, que facilita o controlo sensível e manipulação de temperatura e a taxa de aumento de temperatura e diminuição (0,002 com sensibilidade ° C), coordenado com uma interface de vídeo através de uma rotina de LabVIEW para análise em tempo real. Este sistema pode realizar reprodutíveis taxa controlada experiências que são important para investigar a cinética de interações IBP com gelo. Tais experiências podem resolver vários longo debatidas questões que envolvem o mecanismo de ação do IBPS.

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Número de catálogo / modelo </td><td> Comentários </td></tr><tr><td> Imersão em óleo Tipo B </td><td> Cargille Laboratories </td><td> 16484 </td><td></td></tr><tr><td> Drierite </td><td> WA Hammond Drierite </td><td> 043063 2270g </td><td></td></tr><tr><td> Micro solução de limpeza 90 </td><td> Cole-Parmer </td><td> <a href="http://www.coleparmer.com/Product/Cole_Parmer_small_sup_reg_sup_small_Micro_90_small_sup_reg_sup_small_cleaning_solution_case_of_four_4_Liter_bottles/EW-18100-11" target="_blank">EW-18100-11</a> </td><td></td></tr><tr><td> Capilar extrator </td><td> Narishige </td><td> PB-7 </td><td></td></tr><tr><td> Tubos capilares de vidro </td><td> Marca GnbH Pessoa </td><td> 7493 21 </td><td> 75 mm, 1,15 Diâmetro </td></tr><tr><td> Temperaturacontrolador </td><td> Newport, Irvine, Califórnia, Estados Unidos </td><td> Modelo 3040 </td><td> Modelo 3040 </td></tr><tr><td> Microscópio óptico </td><td> Olimpo </td><td> Modelo BH2 </td><td></td></tr><tr><td> Objetiva de 10x </td><td> Olimpo </td><td></td><td> Plano S 10, 0.3, 160/0.17 </td></tr><tr><td> Objetivo 50x </td><td> Nikon </td><td></td><td> CF plano, 50X/0.55 EPI ELWD </td></tr><tr><td> Câmera CCD </td><td> Provideo </td><td> CVC-140 </td><td></td></tr><tr><td> Tubos de Tygon </td><td> Saint-Gobain, em Paris, França </td><td></td><td> Formulação Tubing Tygon S-50-HL </td></tr><tr><td> Seringa de vidro (2 ml) </td><td> Poulten-Graf, Wertheim, Alemanha </td><td> 7 10227 </td><td></td></tr><tr><td> GPIB-PCI </td><td> National Instruments, Austin, Texas, EUA </td><td> 778032-01 </td><td></td></tr><tr><td> Vídeo quadro grabber IMAQ-PCI-1407 </td><td> National Instruments, Austin, Texas, EUA </td><td> 322156B-01 </td><td></td></tr><tr><td> LabVIEW Software System Design </td><td> National Instruments, Austin, Texas, EUA </td><td> Versão 8 </td><td></td></tr><tr><td> O software DivX Autor </td><td> Divx LLC, San Diego, CA, EUA </td><td></td><td></td></tr></tbody></table>

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).

A medição do tempo de dependência TH O LabVIEW operado osmómetro nanolitro facilita o desempenho de medições precisas de atividade TH. A taxa de redução da temperatura constante permitiu a medição da dependência do tempo TH. O controle preciso da temperatura activado pelo osmómetro nanolitro era crucial para estas experiências. O tempo de exposição de um cristal de gelo para os IBPS em solução é definido como o período de tempo desde a formação do cristal (no final do processo de fusão) até que o crescimento súbito de gelo em volta do cristal (burst cristal). Nós descobrimos que o tempo de exposição dos cristais de gelo para os IBPS crucialmente afectaram a actividade TH. Períodos curtos de exposição ao IBP (alguns segundos), produzida uma actividade TH baixo na solução Mp IBP-GFP (2,4 mM) (Figura 5). A atividade TH aumentou com o tempo de exposição IBP até atingir um patamar de 4 minutos de exposição IBP. A concentrações mais elevadas do IBP, a placaau foi alcançado em menor tempo. <img alt="Figura 1" src="/files/ftp_upload/4189/4189fig1.jpg" /> Figura 1. IBPS diagrama esquemático que ilustra adsorvido em gelo. Aprovada com a permissão de 10. <img alt="Figura 2" fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> Figura 2. A fase de arrefecimento. A) Ligação a tubos no microscópio. B) Sem ligação superior. C) Diagrama esquemático. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig2large.jpg" target="_blank"> </a><img alt="Figura 3" src="/files/ftp_upload/4189/4189fig3.jpg" /> Figura 3. Screenshot da interface do LabVIEW. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">Cl</a>Ick aqui para ver maior figura. <img alt="Figura 4" fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> Figura 4. Gráfico a estabilidade da temperatura. O controlador de temperatura foi ajustado para diminuir a temperatura de 0.01 ° C a cada 15 segundos. <img alt="Figura 5" fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> Figura 5. Mp actividade TH IBP como uma função do tempo de exposição de cristal de gelo com as IBPS. Cada ponto de tempo é a média de 3-6 experiências.

Watch this Scientific Journal Video about LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations at JoVE.com

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Proteínas de ligação de gelo (IBPS), também conhecidas como proteínas anticongelantes, inibir o crescimento do gelo e são um aditivo promissor para uso na criopreservação de tecidos. A principal ferramenta utilizada para investigar IBPS é o osmómetro nanolitro. Nós desenvolvemos uma casa projetada fase de arrefecimento montado em um microscópio óptico e controlados usando uma custom-built rotina LabVIEW. O osmómetro nanolitro descrito aqui manipulado a temperatura da amostra de forma ultra-sensíveis.

LabVIEW operado Osmometer nanolitro Novel de Investigações de gelo ligação às proteínas

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

labview-operated-novel-nanoliter-osmometer-for-ice-binding-protein

Research

JoVE Journal

Chemistry

20.3K visualizações.  The Hebrew University of Jerusalem. Proteínas de ligação de gelo (IBPS), também conhecidas como proteínas anticongelantes, inibir o crescimento do gelo e são um aditivo promissor para uso na criopreservação de tecidos. A principal ferramenta utilizada para investigar IBPS é o osmómetro nanolitro. Nós desenvolvemos uma casa projetada fase de arrefecimento montado em um microscópio óptico e controlados usando uma custom-built rotina LabVIEW. O osmómetro nanolitro descrito aqui manipulado a tempe...

Vídeo: LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

Passivação da superfície de Estudos de proteína única molécula

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

Química

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.

A ligação covalente de BMP-2 em superfícies com uma monocamada auto-montada Abordagem

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 ...

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.

Determinando os planos de ligação de gelo de proteínas anticongelantes pela afinidade do plano de gelo baseado em fluorescência

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 ...

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

Interferometria Bio-layer para Medição Cinética de interações proteína-proteína e alostéricas Ligante Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

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 ...

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.

Emulsões água em óleo: Um novo sistema para a montagem de proteínas de ligação de clorofila solúveis em água com pigmentos hidrofóbicos

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

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

Um ELISA baseada Encadernação e Método de competição de determinar rapidamente interacções ligando-receptor

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

NC-GELFrEE - Limpar nativo de gel eluída fracção líquida Entrapment Electroforese

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

As investigações sobre o Complexo Ga (III) de EOB-DTPA e sua<sup&gt; 68</sup&gt; Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

Novas técnicas para observar a dinâmica estrutural de cristais líquidos de Photoresponsive

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...