Este trabalho foi financiado por doações da Fundação Canadense para a Inovação (CFI), o Instituto Canadense de Pesquisa em Saúde (CIHR), e as Ciências Naturais e Pesquisa de Engenharia Council of Canada (NSERC).

Parte 1: Rompimento da Influenza A Virion <ol><li> Adicionar 750 mL de tampão MNT (20 mM MES, 150 mM NaCl, 30 mM Tris, pH 7,5) em um tubo de centrífuga Beckman policarbonato (11 mm x 34 mm) projetado para caber em um rotor TLA-120,2 para uso em um Optima Beckman Max-E ultracentrífuga. </li><li> Adicionar 500 mL do vírus influenza A (H3N2 X-31 A/AICHI/68 tensão; 2 mg / ml) em que o tubo. Misture o vírus com o buffer MNT pipetando cima e para baixo várias vezes. </li><li> Centrifugar por 10 minutos a 109.000 xg, 4 ° C, em uma centrífuga Beckman Optima Max-E usando um rotor TLA-120,2. </li><li> Remover o sobrenadante e ressuspender o sedimento em 500 mL de buffer interrupção (100 mM KCl, 5 mM MgCl 2, 5% (w / v) de glicerol, 50 octylglucoside mM, 10 mg / ml lysolecithin, 1,5 mM ditiotreitol, 100 mM MES, pH 5,5). </li><li> Vortex vigorosamente, e em seguida, agitar a 31 ° C por 20 minutos em um Thermomixer eppendorf. </li></ol> Parte 2: O glicerol Gradient Centrifugação Velocity Sedimentos <ol><li> Prepare um gradiente de glicerol, colocando as seguintes quantidades de glicerol em um tubo de centrífuga Beckman UltraClear (13 mm x 51 mm): 1 ml de 70% (v / v) de glicerol, 0,75 ml de glicerol 50%, 0,375 ml de glicerol 40%, e 1,8 ml de glicerol 33%. Soluções de glicerol são feitos através da mistura de glicerol puro com tampão NM (150 mM NaCl, 50 mM MES, pH 5,5). Também preparar um tubo de equilíbrio contendo tanto glicerol e buffer. </li><li> Vortex da amostra viral interrompeu novamente, e coloque-os no gradiente de glicerol. </li><li> Centrifugar o gradiente para 3,75 horas em 217.000 xg, 4 ° C, em um MLS-50 Beckman rotor balançando-balde. </li><li> Após a centrifugação é completa, coletar manualmente 250 mL alíquotas do gradiente a partir do topo do tubo. Mantenha frações no gelo ou a 4 ° C. </li></ol> Parte 3: Análise das Frações Gradient Glicerol por SDS Eletroforese em Gel de Poliacrilamida <ol><li> Remover 20 l de cada fração para um novo tubo. </li><li> Adicionar 5 mL de tampão de amostra SDS-PAGE 5x. </li><li> Calor a 95 ° C por 5 minutos. </li><li> Girar as amostras de forma breve e carregá-los em um gel de poliacrilamida 10%, e os marcadores de peso molecular em um dos poços. </li><li> Executar os exemplos sobre o gel até o corante azul de bromofenol carregamento chega perto do fundo do gel. </li><li> Manchar o gel com Coomassie blue. </li></ol> Parte 4: Concentração das frações vRNP <ol><li> Escolha as frações de glicerol contendo principalmente NP, combiná-los e distribuí-los em dois tubos de centrífuga Beckman policarbonato (11 mm x 34 mm). </li><li> Preencher cada tubo com dietil pyrocarbonate (DEPC) tratados com água ultrapura, e pipeta cima e para baixo várias vezes para misturar. </li><li> Centrífuga de 4,5 horas a 157.000 xg, 4 ° C, em um TLA-120,2 Beckman rotor. </li><li> Remover o sobrenadante e ressuspender o sedimento em 50 ul de água tratada DEPC-. </li><li> Se desejar, o 280 A do vRNPs concentrada pode ser medido. Como NP é o principal componente presente nas frações agrupadas, uma estimativa da molaridade de NP pode ser calculada usando seu coeficiente de extinção de 55.350 M -1 cm -1 (como determinado através ProtParam 3). </li><li> Alíquota do vRNPs purificado, e armazená-las congeladas a -80 ° C para uso posterior. </li></ol> Parte 5: Coloração Negativa de vRNPs <ol><li> Diluir 1 ml de vRNP purificada em 9 mL de recém-feitos e filtrada duas vezes-tampão MNT (20 mM MES, 150 mM NaCl, 30 mM Tris, pH 7,5). Buffers outros também são OK para diluir o vRNPs e para lavar a grade de amostra (passo 5.5), mas não use PBS se acetato de uranila é usado para a coloração negativa. </li><li> De descarga luminescente grade um espécime (TEM cobre) previamente revestida com uma película de carbono e parlodion por 30 segundos. </li><li> Segure a grade espécime recém-brilho-alta com uma pinça, e aplicar uma queda de 5 mL de vRNP diluído na grade da amostra. </li><li> Deixe a gota de amostra na grade por 8 minutos. </li><li> Enquanto espera, coloque uma pequena tira de Parafilm no banco, e dispensar 2 gotas contendo 10 ml cada um buffer de MNT (para lavar a grade), e uma queda de 80 mL da solução de coloração feita recentemente (1 molibdato de amônio ou 1% acetato de uranila%) para o Parafilm. </li><li> Após os 8 minutos de adsorção, pavio de parte da solução da amostra a partir da rede amostra com um pedaço de papel de filtro (cortado em triângulos). Não deixe que a grade de amostra para secar. </li><li> Lavar grade amostra em 2 gotas contendo 10 ml cada um buffer de MNT para um tempo total de 1 minuto. Isto é feito com cuidado redução da grade amostra na queda, e depois wicking de parte da solução da amostra a partir da rede amostra com um pedaço de papel de filtro (sem deixar a grade amostra seca). </li><li> Imediatamente após wicking fora a última gota de tampão da rede de espécime, completasubmergir ly grade da amostra dentro da gota da mancha e esperar um minuto. </li><li> Pavio completamente off a solução mancha da rede amostra com um pedaço de papel de filtro. </li><li> Permitir que a grade espécime secarem por alguns minutos antes da observação ao microscópio eletrônico de transmissão. </li></ol> Parte 6: Resultados Representante: <img src="/files/ftp_upload/old/1105_2009_1_22_1/1105_Figure2.png" alt="Figura 2" width="600" height="215" /> Figura 1 Como influenza A NP (~ 56 kDa) é a principal proteína encontrada em complexos ribonucleoproteína viral, a banda NP geralmente é o mais forte da banda nas frações contendo o vRNPs (Figura 1). Além de NP, cada vRNP influenza também contém uma cópia de uma RNA polimerase trimérico (pesos moleculares de kDa 82, 86 e 86,5) complexo. Estes podem ou não ser visíveis por Coomassie coloração azul porque a sua abundância é baixa comparada com a de PN. O polimerases, no entanto, pode ser detectado se, em vez de um azul Commassie mancha, uma mancha de prata do gel é realizada. A proteína de matriz M1 influenza (~ 28 kDa) deve ser minimamente presentes nas frações onde o NP frações pico de proteína estão presentes. <img src="/files/ftp_upload/old/1105_2009_1_22_2/1105_3.png" alt="Figura 3" width="600" height="360" /> Figura 2 VRNPs negativamente manchada como visto sob um microscópio eletrônico de transmissão deve render vRNPs que se assemelham em forma de bastonete partículas com comprimento variável, que são cerca de 30 nm até 120 nm de comprimento (Figura 2a). O NP oligomérica é organizada como uma cadeia de moléculas de NP que é ainda mais dobrada em uma estrutura de dupla hélice repetir, assim loops de ambos os extremos destas partículas em forma de bastonete às vezes pode ser visto (Figura 2b).

<ol>
	<li>Kemler, I., Whittaker, G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+microinjected+influenza+virus+ribonucleoproteins.">Nuclear import of microinjected influenza virus ribonucleoproteins.</a> Virology. 202, 1028-1033 (1994).</li><li>Wu, W. W. H., Weaver, L. L., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+the+nuclear+localization+sequences+on+influenza+a+ribonucleoprotein+complexes.">Ultrastructural analysis of the nuclear localization sequences on influenza a ribonucleoprotein complexes.</a> J Mol Biol. 374, 910-916 (2007).</li><li>Gasteiger, E., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Wu, W. W. H., Sun, Y. H. B., Pant&#233;, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+influenza+A+viral+ribonucleoprotein+complexes+is+mediated+by+two+nuclear+localization+sequences+on+viral+nucleoprotein.">Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein.</a> Virol J. 4, 49-49 (2007).</li><li>Babcock, H. P., Chen, C., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-particle+tracking+to+study+nuclear+trafficking+of+viral+genes.">Using single-particle tracking to study nuclear trafficking of viral genes.</a> Biophys J. 87, 2749-2758 (2004).</li></ol>

A purificação da vRNPs é baseado no procedimento descrito por Kemler et al. (1994). 1 Nós e os outros também têm usado este protocolo para isolar vRNPs para estudar sua importação nuclear. 2,4,5 Recomendamos o uso de RNase dicas e tubos ao manipular vRNPs porque o genoma viral é composto de RNA, e, portanto, degrada facilmente na presença de RNA. Além disso, todos os buffers deve ser feita em água que é RNase livre. O protocolo aqui descrito foi re...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>H3N2 X-31 A/AICHI/68 influenza A</td>
<td>&nbsp;</td>
<td>Charles River Laboratories</td>
<td>490715</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M-8250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G33-1</td>
<td>&nbsp;</td>
<tr>
<td>Octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O-8001</td>
<td>&nbsp;</td>
<tr>
<td>Lysolecithin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-4129</td>
<td>&nbsp;</td>
<tr>
<td>Dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-9779</td>
<td>&nbsp;</td>
<tr>
<td>Coomassie brilliant blue G-250</td>
<td>&nbsp;</td>
<td>Kodak</td>
<td>1367796</td>
<td>&nbsp;</td>
<tr>
<td>diethyl pyrocarbonate (DEPC)-treated water</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>750024</td>
<td>&nbsp;</td>
<tr>
<td>Uranyl Acetate</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>19481</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Molybdate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A-674</td>
<td>&nbsp;</td>
<tr>
<td>Optima MAX-E Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>434491</td>
<td>&nbsp;</td>
<tr>
<td>MLS-50 Rotor Package, Swinging Bucket</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>367280</td>
<td>&nbsp;</td>
<tr>
<td>TLA-120.2 Rotor Assembly, Fixed-Angle, Titanium</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362046</td>
<td>&nbsp;</td>
<tr>
<td>Eppendorf Thermomixer</td>
<td>&nbsp;</td>
<td>Brinkman</td>
<td>022670000</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

purification and visualization of influenza a viral ribonucleoprotein complexes

A influenza A genoma viral é composto por oito sentido negativo, as moléculas de RNA de fita única, embalados individualmente com várias cópias da influenza A nucleoproteína (NP) em partículas ribonulceoprotein viral (vRNPs). O vRNPs influenza são colocados dentro do envelope viral. Durante a entrada de celulares, no entanto, esses complexos vRNP são liberados para o citoplasma, onde eles ter acesso ao equipamento de transportes anfitrião nuclear. A fim de estudar a importação nuclear de vRNPs influenza ea replicação do genoma da gripe, é útil para trabalhar com vRNPs isolada de modo que outros componentes do vírus não interferem com estes processos. Aqui, descrevemos um procedimento para purificar essas vRNPs do vírus influenza A. O procedimento começa com o rompimento da influenza A virion com detergentes, a fim de liberar os complexos vRNP do virion envolvido. O vRNPs são então separados dos outros componentes da influenza A virion em um gradiente de glicerol 33-70% descontínua por sedimentação velocidade. As frações obtidas a partir do gradiente de glicerol são analisados ​​na via SDS-PAGE após coloração com Coomassie blue. As frações de pico contendo NP são então reunidos e concentrados por centrifugação. Após a concentração, a integridade do vRNPs é verificada através da visualização do vRNPs por microscopia eletrônica de transmissão após coloração negativa. A purificação do gradiente de glicerol é uma modificação do que a partir de Kemler<em&gt; Et al.</em&gt; (1994)<sup&gt; 1</sup&gt;, Ea coloração negativa foi realizado por Wu<em&gt; Et al.</em&gt; (2007).<sup&gt; 2</sup

Watch this Scientific Journal Video about Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes at JoVE.com

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

O genoma do vírus influenza A é composto por oito complexos separada de RNA e proteínas, denominadas complexos de ribonucleoproteínas viral (vRNPs). Este artigo descreve a purificação gradiente glicerol e transmissão de visualização microscopia eletrônica de influenza A vRNPs.

Purificação e Visualização de Influenza A complexos de ribonucleoproteínas Viral

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

purification-visualization-influenza-viral-ribonucleoprotein

Research

JoVE Journal

Biology

12.9K visualizações.  University of British Columbia - UBC. O genoma do vírus influenza A é composto por oito complexos separada de RNA e proteínas, denominadas complexos de ribonucleoproteínas viral (vRNPs). Este artigo descreve a purificação gradiente glicerol e transmissão de visualização microscopia eletrônica de influenza A vRNPs.

Vídeo: Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Biologia

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Purificação de preparação, e Caracterização de Complexos de lantanídeos para uso como agentes de contraste para Imagem por Ressonância Magnética

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Purificação da Hsp104, um Disaggregase Protein

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificação cromatográfica de Ribossomos Yeast Highly Active

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Método para o isolamento e identificação de mRNAs, microRNAs e componentes proteicos de complexos de ribonucleoproteínas de extratos celulares utilizando RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Mapeamento baseado em AFM das propriedades elásticas de paredes celulares: em tecido, os celulares, e as Resoluções Subcelulares

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Rápido isolamento e purificação de mitocôndrias para o transplante pela dissociação do tecido e Filtração Diferencial

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Geração e Purificação de INO80 cromatina remodelação Complexos Humanos e subcomplexos

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

A validação de um modelo de camundongo para Disrupt Complexos LINC de um modo específico de célula

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...