Siamo grati a Frank Ervin, Roberta Palmour e il personale delle scienze comportamentali Laboratori Fondazione situato a St Kitts, nelle Indie Occidentali, per il supporto continuo del nostro lavoro primati. Questo lavoro è stato sostenuto da una borsa di studio Fragile X Research Foundation of Canada (FXRFC) a SZ e - sovvenzioni di funzionamento dal Canadian Institutes of Health Research (AC) e il National Engineering and Research Council del Canada (MP).

Immunohistochemisty è una delle tecniche più utilizzate per la caratterizzazione di espressione della proteina nel cervello di vari modelli sperimentali animali. E &#39;relativamente facile da condurre sistematiche procedure di immunoistochimica sul cervello di roditori e di altri comuni modelli sperimentali con le dimensioni del cervello simili. Tuttavia, non c&#39;è lavoro pubblicato a nostra conoscenza, che fornisce un resoconto completo di come svolgere le procedure di immunolocalizzazione tale attraverso un intero cervello della scimmia. Quello che segue è una descrizione dettagliata di come preparare un intero cervello di scimmia per la grande rivelazione immunoistochimica di proteine ​​target diversi. Questo lavoro è emerso come un risultato della collaborazione tra sforzi commerciali e accademici. Come tale, i dettagli relativi al tessuto embedding e sezionamento rimangono una conoscenza di proprietà di NSA. Parte 1: trattamento degli animali e di preparazione dei tessuti Il cervello di una scimmia adulta cercopiteco (Cercopithecus aethiops) è utilizzato per il protocollo attuale. Tutte le procedure vengono eseguite nel rispetto della Canadian Council sulla cura degli animali (CCAC) le linee guida per l&#39;uso e la cura degli animali nella ricerca biomedica 1. <ol><li> Animale è profondamente sedato con cloridrato di ketamina (10 mg / kg, im), eutanasia con una dose eccessiva di sodio pentobarbital (25 mg / kg, iv) e perfusi transcardially con 0,1 M PBS fino a completo dissanguato. </li><li> Questa è seguita da una soluzione di paraformaldeide al 4% in PBS per 5 min (~ 1 litro). </li><li> Il cervello esternato è posto in graduato PBS-buffered soluzioni di saccarosio (10, 20 e 30%, in sequenza) che contiene sodio azide 0,02% e mantenuto a 4 ˚ C fino a quando il cervello scende sul fondo del contenitore. La soluzione è sostituita ogni pochi giorni fino a quando il cervello subisce congelamento sezionamento. </li><li> Il cervello viene inviato agli associati Neuroscienze (NSA, Knoxville, TN) da sottoporre ai loro proprietari MultiBrain ™ inclusione e di gelo-sezionamento della tecnologia. </li><li> Sette (7) punti di riferimento di allineamento vengono inseriti nella matrice embedding in modo che le sezioni possono essere orientati correttamente (vale a dire, sinistra-destra di orientamento) e ri-allineato dopo l&#39;elaborazione istologica è completa. </li><li> Il blocco congelato è fotografato digitalmente il volto blocco prima di ogni sezione seriale viene raccolto dal blocco. Le immagini digitali così come sezioni da questo cervello può essere utilizzato successivamente per la ricostruzione 3D delle mappe anatomiche ed istologiche, rispettivamente. </li><li> Seriale sezioni coronali libero di fluttuare in uno spessore di 50 micron per sezione, sono raccolti presso l&#39;intero cervello. </li></ol> Parte 2: elaborazione istologica Le sezioni sono scelti in un dato intervallo spaziale (per esempio, 500-micron) e per i nostri scopi sperimentali sono stati elaborati con i seguenti anticorpi:-X fragile proteina ritardo mentale (FMRP; Chemicon, Temecula, CA), SMI32 (Sternberger monoclonali Inc.; Baltimore, MD), e NeuN (Chemicon, Temecula, CA). FMRP è una proteina citoplasmatica che trovano abbondantemente nei neuroni del cervello vettore normale e permutazione 2. NeuN (Nuclei neuronale) riconosce espressamente il legame al DNA dei neuroni specifici NeuN proteina che è presente nella maggior parte dei neuroni e si distribuisce nei nuclei neuronali, perikarya e alcuni processi neuronali prossimale 3. SMI-32 è un anticorpo monoclonale che riconosce la non-fosforilata epitopo su proteine ​​neurofilamenti 4. <ol><li> Ad un intervallo di 500 micron, circa 140 sezioni vengono utilizzati per anticorpi. </li><li> Tutti, lava incubazioni e passi colorazione sono svolte con agitazione mite grazie ad antenne colorazione specializzati e canestri (HistoTools, Knoxville, TN). </li><li> Le sezioni sono prima incubate in una soluzione contenente 0,1 M PBS/0.3% Triton X-100 (TX) / 5% di siero normale roca (SSN) per 60 minuti al fine di ridurre legame aspecifico di molecole di anticorpi e la colorazione di fondo che ne derivano. </li><li> Le sezioni contrassegnate per immunocolorazione FMRP subire un ulteriore passo di recupero dell&#39;antigene utilizzando Unmasking Solution Antigen (Labs Vector, Burlingame, CA) secondo le istruzioni del fornitore. </li><li> Le sezioni sono poi incubate durante la notte e in giorni distinti nelle loro soluzioni di anticorpi rispettivi contenente 0,1 M PBS/0.3% TX / 3% NHS (con un fattore di diluizione di 1 / 5, 000 per FMRP e SMI-32 e 1 / 2, 000 per gli anticorpi NeuN. </li><li> Il giorno successivo, le sezioni vengono lavate per tre periodi di 10 minuti in soluzione di lavaggio (0,1 M PBS/0.3% TX), seguita da incubazione per 2 ore a temperatura ambiente in biotinilato anti-topo secondario cresciuto a cavallo (Vector Labs, 1 / 1.000 diluizione in 0,1 M PBS/0.3% NHS TX / 3%). </li><li> Dopo un ulteriore insieme di 3 10 minuti lava, le sezioni sono poste in una soluzione di avidina-biotina coniugato complesso perossidasi (Vector Labs) per 1 ora a temperatura ambiente. </li><li> Infine, dopo un&#39;altra serie di lavaggi, le sezioni sono sottoposti per 10 min fino ad un diametro maggiore di nichelminobenzidine (Ni-DAB), reazione che produce una macchia blu scuro all&#39;interno dei neuroni immunoreattive. </li><li> Al completamento della procedura istologici, le sezioni sono montati su gelatina rivestita vetrini. </li><li> Tutte le sezioni sono dotate di aria secca durante la notte e coperti secondo le procedure standard. </li></ol> Parte 3: Risultati Rappresentante: Questo metodo produce un profilo completo espressione di una proteina bersaglio di interesse in tutta l&#39;intero cervello della scimmia. Qui vi mostriamo rappresentante sezioni coronali che forniscono un&#39;istantanea di FMRP, NeuN e SMI32 espressione nel cervello della scimmia stessa. <img src="/files/ftp_upload/1286/1286fig1.png" alt="" /> Figura 1: piatto di colorazione (A) e basket (B), utilizzato su larga scala l&#39;elaborazione in batch di free-floating sezioni per immunolocalizzazione. <img src="/files/ftp_upload/1286/1286fig2.png" alt="" /> Figura 2: Rappresentante sezioni coronali da un cervello di scimmia cercopiteco colorate per FMRP (A), NeuN (B) e SMI32 (C) gli anticorpi.

<ol>
	<li>Olfert, E. D., Cross, B. M., McWilliam, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to the Care and Use of Experimental Animals. , (1993).</li><li>Devys, D., Lutz, Y., Rouyer, N., Bellocq, J. P., Mandel, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FMR-1+protein+is+cytoplasmic,+most+abundant+in+neurons+and+appears+normal+in+carriers+of+a+fragile+X+premutation.">The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation.</a> Nat Genet. 4, 335-340 (1993).</li><li>Mullen, R. J., Buck, C. R., Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuN,+a+neuronal+specific+nuclear+protein+in+vertebrates.">NeuN, a neuronal specific nuclear protein in vertebrates.</a> Development. 116, 201-211 (1992).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proc Natl Acad Sci U S A. 80, 6126-6130 (1983).</li></ol>

The authors have nothing to disclose.

Ci sono due passi fondamentali in questa procedura che rendono grande il rilevamento di proteine ​​in un cervello della scimmia intero possibile. Uno è il protocollo di inclusione e di sezionamento, che rimane la conoscenza di proprietà della NSA. L&#39;altra è l&#39;uso di piatti colorazione e cesti forniti da HistoTools. Quest&#39;ultimo consente di utilizzo semplice e veloce di molti (~ 40) sezioni in un dato momento. Inoltre fornisce i mezzi per trattare in modo uniforme tutte le sezioni in tutto il cervello e fa per un trattamento scientificamente suono istologici. Inoltre, l&#39;uso di punti di riferimento incorporato offre il vantaggio aggiunto di essere in grado di acquisire digitalmente ai modelli di marcatura da diapositive essiccate e coperti e di sottoporre i file digitalizzati a varie forme di analisi. Anche se ci forniscono esempi di tre anticorpi, questa procedura può essere applicata a una vasta gamma di altre proteine ​​bersaglio così come le macchie istologiche, in particolare quelli che rivelano le aree corticali citoarchitettura diversi e, quindi, utilizzato per scopi di mappatura.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>anti-FMRP monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB2160</td>
<td>Requires antigen retrieval in fixed tissue.</td>
</tr>
<tr>
<td>anti-NeuN monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB377</td>
<td>N/A</td>
</tr>
<tr>
<td>anti-Neurofilament H monoclonal antibody</td>
<td>&nbsp;</td>
<td>Sternberger Monoclonals Inc.</td>
<td>SMI32</td>
<td>N/A</td>
</tr>
<tr>
<td>Antigen Unmasking Solution</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-3300</td>
<td>N/A</td>
</tr>
<tr>
<td>Normal horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050122</td>
<td>500 mL</td>
</tr>
<tr>
<td>Biotinylated Anti-Mouse IgG (H+L), made in horse</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>BA-2000</td>
<td>&nbsp;</td>
<tr>
<td>VECTASTAIN ABC Kit (Standard)</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>PK-4000</td>
<td>1.5 mg</td>
</tr>
<tr>
<td>Staining dish for floating sections</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10009</td>
<td>12 x 65 mm</td>
</tr>
<tr>
<td>Staining transport basket</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10012</td>
<td>large 
(fits 12 x 65 mm dish)</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>P8514B</td>
<td>N/A</td>
</tr>
<tr>
<td>DAB</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>AC11209-0050</td>
<td>N/A</td>
</tr>		</tbody>
		</table>
		</div>

batch immunostaining for large-scale protein detection in the whole monkey brain

Immunoistochimica (IHC) è una delle tecniche di laboratorio più utilizzato per la rilevazione di proteine ​​bersaglio<em&gt; In situ</em&gt;. Le questioni concernenti il ​​pattern di espressione di una proteina bersaglio attraverso tutto il cervello è relativamente facile rispondere quando si usa IHC nei cervelli piccoli, come quelli dei roditori. Tuttavia, rispondere alle stesse domande nel cervello di grandi dimensioni e contorto, come quelli dei primati presenta una serie di sfide. Qui vi presentiamo un approccio sistematico per immunolocalizzazione di proteine ​​bersaglio in un cervello di scimmia adulta. Questo approccio si basa sul tessuto inclusione e il sezionamento metodologia di Neuroscienze Associates (NSA), oltre a strumenti sviluppati appositamente per batch-colorazione di free-floating sezioni. Essa si traduce in colorazione uniforme di una serie di sezioni che, ad un intervallo particolare, rappresenta l&#39;intero cervello. Le sezioni colorate risultante può essere sottoposto a una vasta gamma di procedure analitiche per misurare i livelli di proteina, la popolazione di neuroni che esprimono una certa proteina.

Watch this Scientific Journal Video about Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain at JoVE.com

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Su larga scala immunolocalizzazione di proteine ​​bersaglio attraverso tutto il cervello dei primati è possibile utilizzando tessuti romanzo di inclusione e di sezionamento metodi combinati con l&#39;impiego di attrezzature creativo per la colorazione in batch di più liberamente fluttuanti sezioni in un dato momento.

Immunostaining lotto per la grande rivelazione di proteine ​​nel cervello della scimmia Whole

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

batch-immunostaining-for-large-scale-protein-detection-whole-monkey

Nanyang Technological University

Research

JoVE Journal

Biology

12.6K Views.  Montreal Neurological Institute. Su larga scala immunolocalizzazione di proteine ​​bersaglio attraverso tutto il cervello dei primati è possibile utilizzando tessuti romanzo di inclusione e di sezionamento metodi combinati con l'impiego di attrezzature creativo per la colorazione in batch di più liberamente fluttuanti sezioni in un dato momento.

Video: Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Su larga scala Schermi di librerie metagenomiche

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Ricerca

Biologia

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Rilevamento di proteine ​​Ubiquitinazione

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purificazione della M. magneticum Strain AMB-1 proteina Magnetosome Associated MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Screen piccole molecole

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium Assay per i grandi SNP genotipizzazione Applicazioni

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Una strategia per sensibili, su larga scala quantitative Metabolomica

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Indagare interazioni proteina-proteina in cellule vive utilizzando il trasferimento bioluminescenza Resonance Energy

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: Metodo su vasta scala per la quantificazione multiparametrica della crescita del lievito

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identificazione di nuovi regolatori della traspirazione vegetale mediante screening termico su larga scala in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Immunostaining lotto per la grande rivelazione di proteine nel cervello della scimmia Whole

Riepilogo

Esplora Altri Video

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Su larga scala Schermi di librerie metagenomiche

Rilevamento di proteine Ubiquitinazione

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

Purificazione della M. magneticum Strain AMB-1 proteina Magnetosome Associated MamAΔ41

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Infinium Assay per i grandi SNP genotipizzazione Applicazioni

Una strategia per sensibili, su larga scala quantitative Metabolomica

Indagare interazioni proteina-proteina in cellule vive utilizzando il trasferimento bioluminescenza Resonance Energy

ODELAY: Metodo su vasta scala per la quantificazione multiparametrica della crescita del lievito

Identificazione di nuovi regolatori della traspirazione vegetale mediante screening termico su larga scala in Helianthus Annuus

Immunostaining lotto per la grande rivelazione di proteine ​​nel cervello della scimmia Whole

Riepilogo

Esplora Altri Video

Capitoli in questo video

Su larga scala Schermi di librerie metagenomiche

Rilevamento di proteine ​​Ubiquitinazione

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

Purificazione della M. magneticum Strain AMB-1 proteina Magnetosome Associated MamAΔ41

Large Scale Zebrafish-Based In vivo Screen piccole molecole

Infinium Assay per i grandi SNP genotipizzazione Applicazioni

Una strategia per sensibili, su larga scala quantitative Metabolomica

Indagare interazioni proteina-proteina in cellule vive utilizzando il trasferimento bioluminescenza Resonance Energy

ODELAY: Metodo su vasta scala per la quantificazione multiparametrica della crescita del lievito

Identificazione di nuovi regolatori della traspirazione vegetale mediante screening termico su larga scala in Helianthus Annuus

Immunostaining lotto per la grande rivelazione di proteine nel cervello della scimmia Whole

Rilevamento di proteine Ubiquitinazione