Name: antibody labeling with fluorescent dyes using magnetic protein a and protein g beads
Uploaded: 2016-09-15T14:00:00.000+00:00
Duration: 6 min 48 s
Description: Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

1. Captura de Anticorpos em G Beads Alta Capacidade Protein Magnetic A ou a proteína Magnetic <ol><li> Uniformemente re-suspender as esferas magnéticas por agitação suave. Mantenha o uniforme de suspensão quando alíquotas esferas. </li><li> Adicionar 50 ul de suspensão de esferas para um tubo de microcentrífuga de 1,5 ml. Coloque no suporte magnético por 10 s. Retire cuidadosamente o tampão de armazenamento. </li><li> Adicionar 250 ul de tampão de ligação do anticorpo. </li><li> Misture e coloque no suporte magnético por 10 s. Retire cuidadosamente o tampão de ligação. </li><li> Adicionar 1,0 ml de amostra contendo 50-100 ng de anticorpo com as esferas. As amostras podem ser purificados anticorpos ou anticorpos no meio celular. </li><li> Misturar amostras durante 60 min à temperatura ambiente usando um misturador de vórtice ou misturador-sobre-extremidade final. </li><li> Coloque tubo no suporte magnético por 10 seg e remover o sobrenadante. </li><li> Adicionar 250 ul de anticorpo tampão de ligação / lavagem e misturar. Coloque no suporte magnético por 10 seg e remover o tampão de ligação / lavagem. Repita este step para um total de duas lavagens. </li><li> Vá para a Seção 2 a anticorpos etiqueta utilizando química amina ou a Seção 3 para anticorpos etiqueta utilizando química tiol. </li></ol> 2. Rotulagem de anticorpos utilizando a amina Química <ol><li> conjugado anticorpo <ol><li> Adicionar 100 ul de tampão de conjugação de amina para os grânulos. </li><li> Dissolver os corantes fluorescentes reactivos com aminas (AlexaFluor 532-SE) a 10 mg / ml por adição de 100 ul de dimetilsulfóxido (DMSO) a 1,0 mg de corante. Misture em vortex. Faça esta solução imediatamente antes da utilização. </li><li> Adicionar 2,5 mL de corante reactivo a amina de 100 ug de anticorpo. Nota: normalmente um excesso molar de 5-20 de corante reactivo é recomendado. No entanto, a quantidade de corante reactivo adicionado à reacção tem de ser empiricamente optimizadas dependendo do corante desejado rácio de anticorpos e propriedades intrínsecas de anticorpos. </li><li> Misturar amostras durante 60 min à temperatura ambiente usando um misturador de vórtice ou misturador-sobre-extremidade final. Certifique-se que as contas permanecem em suspension. </li><li> Coloque tubo no suporte magnético por 10 seg e remover o sobrenadante. </li><li> Adicionar 250 ul de anticorpo tampão de ligação / lavagem e misturar. Coloque no suporte magnético por 10 s. Remova e descarte tampão de ligação / lavagem. Repetir esta etapa para um total de duas lavagens. </li></ol></li><li> Recuperação de anticorpo <ol><li> Adicionar 50 ul de tampão de eluição para os grânulos. </li><li> Mistura-se durante 5 min à temperatura ambiente usando um misturador de vórtice ou misturador-sobre-extremidade final. Certifique-se que as contas permanecem em suspensão. </li><li> Coloque tubo no suporte magnético por 10 s. Remover a amostra e eluiu-se a transferência para um tubo de micro-centrifugação novo contendo 5 ul de tampão de neutralização. </li><li> Repetir o processo mais uma vez e reunir as amostras eluídas. </li><li> Quantificar a concentração de anticorpos e tingir-se anticorpo rácio conforme descrito na Seção 4. </li></ol></li></ol> 3. Anticorpo de marcação utilizando química Tiol <ol><li> Redução de anticorpo <ol><li> Adicionar 250 ulde tampão de conjugação de tiol e mistura. Colocar o tubo no suporte magnético durante 10 seg. Retirar e descartar o buffer. Repita esta etapa duas vezes. </li><li> Adicionar 100 ul de tampão de conjugação de tiol. </li><li> Adicionar ditiotreitol (DTT) até uma concentração final de 2,5 mM. </li><li> Misturar amostras durante 60 min à temperatura ambiente usando um misturador de vórtice ou misturador-sobre-extremidade final. Certifique-se que as contas permanecem em suspensão. </li><li> Colocar o tubo no suporte magnético durante 10 seg e descartar o tampão. </li><li> Adicionar 250 ul de tampão de conjugação de tiol e misturar. Colocar o tubo no suporte magnético durante 10 seg. Retirar e descartar o buffer. Repetir esta etapa para um total de duas lavagens. </li><li> Adicionar 100 ul de tampão de conjugação de tiol. </li></ol></li><li> conjugado anticorpo <ol><li> Dissolve-se o corante reactivo com tiol a 10 mg / ml por adição de 100 ul de DMSO a 1,0 mg de corante. Misture em vortex. Faça esta solução imediatamente antes da utilização. </li><li> Adicionar 2,5 mL de corante reactivo com tiol de 100 ug de anticorpo. Nota: normalmente um excesso molar de 5-20 de corante reactivo é recomendado. No entanto, a quantidade de corante reactivo adicionado à reacção tem de ser empiricamente optimizadas dependendo do corante desejado rácio de anticorpos e propriedades intrínsecas de anticorpos. </li><li> Misturar amostras durante 60 min à temperatura ambiente usando um misturador de vórtice ou misturador-sobre-extremidade final. Certifique-se que as contas permanecem em suspensão. </li><li> Coloque o tubo no suporte magnético por 10 seg e remover o sobrenadante. </li><li> Adicionar 250 ul de tampão de conjugação de tiol e misturar. Coloque no suporte magnético por 10 segundos e retire o tampão. Repetir esta etapa para um total de duas lavagens. </li></ol></li><li> eluto Antibody <ol><li> Adicionar 50 ul de tampão de eluição para os grânulos. </li><li> Mistura-se durante 5 min à temperatura ambiente usando um misturador de vórtice ou misturador-sobre-extremidade final. Certifique-se que as contas permanecem em suspensão. </li><li> Coloque tubo no suporte magnético por 10 s. Remover amostra eluída e transferir para um tubo de micro-centrífuga nova contendo 5 ul de neuttampão ralization. </li><li> Repetir o processo mais uma vez e reunir as amostras eluídas. </li><li> Quantificar a concentração de anticorpos e tingir-se anticorpo rácio conforme descrito na Seção 4. </li></ol></li></ol> Rácio 4. Calcule Dye-to-Antibody <ol><li> Medir a absorvância do conjugado anticorpo-corante a 280 nm (A280) e a ^ max para o corante (Amax). </li><li> Calcula-se a concentração de anticorpo: Concentração de anticorpo (mg / ml) = A 280 - (A máx × CF) / 1.4 em que CF = factor de correcção do corante (fornecido pelo fabricante). </li><li> Calcular a proporção de corante-de-anticorpo: Tintura-se Anticorpo Ratio (DAR) = (A máx 150000 ×) / Ab Concentração (mg / ml) × ε corante onde, corante ε = o coeficiente de extinção do corante no seu máximo de absorvância e o peso molecular do anticorpo = 150000 Da. </li></ol>

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Authors are employees of Promega Corporation.

O objectivo deste estudo foi o de desenvolver um método para marcar anticorpos, presentes no meio celular em baixas concentrações, com uma variedade de moléculas pequenas. Um tal processo permitirá que um grande número de anticorpos, durante as fases iniciais da descoberta de anticorpos, para ser marcado com moléculas pequenas e rastreados utilizando um ensaio de biologicamente relevante. Um desses ensaios é o ensaio de anticorpo de internalização mediada pelo receptor onde internalização pode variar entre o...

Os anticorpos marcados com moléculas pequenas são, talvez, os reagentes mais utilizados na biologia 1,2. Os anticorpos marcados com corantes fluorescentes e biotina são amplamente utilizados em imagiologia, imunoensaios, citometria de fluxo, Western blot, imunoprecipitação e entre outras aplicações 3-6. Anticorpos radiomarcados 3,7 encontrar o uso extensivo em imagiologia e terapia, anticorpos marcados com drogas citotóxicas (ADCs) estão oferecendo novas opções para o tratamento de cancros, e dois ADCs já foram aprovados para uso terapêutico 8. Apesar de sua ampla utilização, os métodos para detecção de anticorpos rotulagem continuam surpreendentemente inalterado e normalmente envolvem múltiplas reações e dessalinização os passos 9-12. métodos de solução funciona muito bem nos casos em que apenas alguns anticorpos necessita ser etiquetado e estão disponíveis em forma altamente purificada em concentração elevada e em volumes suficientes. No entanto, para aplicações mais recentes, como os ADCs, existe umprecisa para marcar anticorpos de hibridoma na fase precoce de modo que eles podem ser rastreados para propriedades biologicamente relevantes, por exemplo, mediada por receptor de anticorpos de internalização 13-16. Na fase de hibridoma, os volumes de amostra são limitados, os anticorpos são expressas em baixas concentrações e uma série de amostras são grandes, portanto, solução baseada em métodos de marcação não são adequados. Para simplificar e melhorar o rendimento dos métodos de rotulagem de anticorpos tradicionais, algumas abordagens alternativas têm sido propostas 17,18. Uma abordagem é a utilização de proteínas de não-magnético grânulos de afinidade embalados em pequenas colunas para capturar anticorpos, seguido pela reacção de marcação e a eluição da proteína marcada e purificada. Este método pode ser utilizado para rotular os anticorpos directamente a partir de meios de células, no entanto, o uso de colunas pode ser trabalhoso. Processo baseado em esférulas magnéticas tem sido relatado recentemente que 19 elimina o uso de colunas e melhora o rendimento mas devidopara o anticorpo limitada capacidade dos grânulos de ligação, apenas a nanograma a baixas quantidades de micrograma de os anticorpos podem ser marcados. Nós recentemente desenvolvido e utilizado alta capacidade magnética Proteína A e Proteína G grânulos (&gt; 20 mg de IgG humana / mL de esferas de liquidados) para rotular anticorpos presentes nos meios de células com moléculas pequenas 20. A elevada capacidade dos grânulos permite dezenas a centenas de microgramas de anticorpo a ser marcado convenientemente e a resposta magnética rápida dos grânulos simplifica o manuseamento e processamento de um grande número de amostras em paralelo. Utilizando corantes fluorescentes como substitutos para moléculas pequenas, que mostram que o método é compatível com a amina e tiol rotulagem química e oferece recuperações elevadas de anticorpos marcados e muito puras. Este protocolo eo vídeo que a acompanha descrevem rotulagem on-gota de anticorpos de ratinho presentes nos meios de células usando G esferas de Proteína Magnetic A e proteína. O protocolo édividida em quatro secções: uma secção descreve a captura de anticorpos para o grânulo a partir de amostras biológicas. Após a captura, a rotulagem de anticorpos com corante fluorescente usando química amina ou utilizando a química do tiol é descrito nas Secções 2 e seção 3, respectivamente. Finalmente, a seção 4 descreve o método para o cálculo da concentração de anticorpos e o corante à relação de anticorpos.

<table><tbody><tr><td>Magne Protein A Beads</td><td>Promega Corporation</td><td>&amp;nbsp;G8781</td><td></td></tr><tr><td>Magne Protein G Beads</td><td>Promega Corporation&amp;nbsp;</td><td>&amp;nbsp;G7471</td><td></td></tr><tr><td>AlexaFluor 532-SE (Succinimidyl Ester)</td><td>Life Technologies</td><td>A20001</td><td></td></tr><tr><td>AlexaFluor 532-ME (Malemide)</td><td>Life Technologies</td><td>A10255</td><td></td></tr><tr><td>AlexaFluor 647-ME (Maleimide)</td><td>Life Technologies</td><td>A20347</td><td></td></tr><tr><td>Fluorescein-ME (Maleimide)</td><td>Life Technologies</td><td>F-150</td><td></td></tr><tr><td>Magnetic Stand</td><td>Promega</td><td>Z5332</td><td></td></tr></tbody></table>

antibody labeling with fluorescent dyes using magnetic protein a and protein g beads

Anticorpos marcados com pequenas moléculas como corantes fluorescentes, medicamentos citotóxicos e traçadores radioativos são ferramentas essenciais na investigação biomédica, imunodiagnóstico e, mais recentemente como agentes terapêuticos. Os métodos tradicionais de anticorpos de rotulagem com pequenas moléculas requerem anticorpos purificados na concentração relativamente elevada, envolvem múltiplos passos de diálise e tem produção limitada. No entanto, várias aplicações, incluindo o campo de Anticorpo drogas conjugados (ADCs), irão beneficiar de novos métodos que permitam a rotulagem de anticorpos diretamente da mídia de celulares. Tais métodos podem permitir que os anticorpos a serem rastreados em ensaios biologicamente relevantes, por exemplo, o ensaio de anticorpo de internalização mediada pelo receptor no caso de ADCs. Aqui, nós descrevemos um (método a-bead) método que permita a marcação de pequenas quantidades de anticorpos directamente a partir de meio celular. Esta abordagem utiliza alta capacidade G esferas de afinidade Protein magnética A e proteína para capturar anticorposa partir dos meios celulares, seguido de marcação com moléculas pequenas utilizando quer química de amina ou de tiol e a subsequente eluição dos anticorpos marcados. Tomando corantes fluorescentes como substitutos para moléculas pequenas, que demonstram a rotulagem sobre-cordão de três diferentes anticorpos de ratinho directamente a partir de meios celulares utilizando tanto amina e química rotulagem tiol. A elevada afinidade de ligação de anticorpos à proteína A e proteína G assegura recuperações elevadas, bem como elevada pureza dos anticorpos marcados. Além disso, a utilização de esferas magnéticas permite que amostras múltiplas para serem manuseados manualmente, melhorando desse modo significativamente a taxa de transferência de rotulagem.

Um esquema para a marcação de anticorpos com moléculas pequenas utilizando G grânulos alta capacidade Proteína magnética A e proteína é mostrada na Figura 1. Os anticorpos capturados sobre a proteína magnética grânulos G podem ser marcados com moléculas pequenas, por exemplo, corantes fluorescentes, utilizando quer química de amina que rotula aminas primárias de ácidos aminados ou da lisina utilizando química de tiol, o qual etiquetas nos tióis reduzidos...

Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

O método no grânulo para a marcação de anticorpos com pequenas moléculas permite a marcação de uma pequena quantidade de anticorpos directamente a partir de meio celular. Este método é compatível com a química de amina e tiol, e pode lidar com várias amostras em paralelo, manualmente ou por meio de plataformas automatizadas.

Rotulagem de anticorpos com corantes fluorescentes utilizando proteína Magnetic A e esferas de proteína G

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

antibody-labeling-with-fluorescent-dyes-using-magnetic-protein

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JoVE Journal

Immunology and Infection

11.8K visualizações.  Promega Corporation.  O método no grânulo para a marcação de anticorpos com pequenas moléculas permite a marcação de uma pequena quantidade de anticorpos directamente a partir de meio celular. Este método é compatível com a química de amina e tiol, e pode lidar com várias amostras em paralelo, manualmente ou por meio de plataformas automatizadas. 

Vídeo: Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Eficiente e Rotulagem de anticorpos por cicloadição Azide-alcino promoveu-Strain Site-specific

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Bioquímica

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Protocolo experimental para Detectar<em&gt; Cianobactérias</em&gt; Em amostras líquidas e sólidas com um Microarray Chip Antibody

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Ambiente

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Rotulagem diferencial de superfície celular e internalizado Proteínas após Anticorpo Alimentação da Vivo Cultivadas neurônios

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Neurociência

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolamento de proteínas lábeis Multi-Complexos por In vivo Controlled Cellular Cross-Linking and Imuno-magnética cromatografia de afinidade

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Biologia

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - um método para identificar em toda a Transcriptoma Sítios de Ligação de Proteínas RNA Binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

Visualização de miniSOG Proteínas Tagged reparo de DNA em combinação com Electron espectroscópica Imagem (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Perfil de expressão gênica específica do tipo de célula no fígado do mouse

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Detecção de Agregação de Proteínas usando Espectroscopia de Correlação de Fluorescência

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Source: Blanco, Y. et al., <a href="https://app.jove.com/v/54994">Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct spots.

Rotulagem de anticorpos com corantes fluorescentes utilizando proteína Magnetic A e esferas de proteína G

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