Wir bedanken uns bei Frank Ervin, Roberta Palmour und das Personal des Behavioural Sciences Foundation Laboratories in St Kitts, West Indies, für ihre anhaltende Unterstützung unserer Primaten Arbeit befindet. Betriebskostenzuschüsse von der Canadian Institutes of Health Research (AC) und der National Engineering and Research Council of Canada (MP) - Diese Arbeit wurde durch ein Stipendium Fragile X Research Foundation of Canada (FXRFC) auf SZ und unterstützt.

Immunohistochemisty ist eine der am häufigsten verwendeten Techniken zur Charakterisierung von Protein-Expression im Gehirn von verschiedenen experimentellen Tiermodellen. Es ist relativ leicht zu systematischen immunhistochemischen Verfahren auf die Gehirne der Nagetiere und andere gemeinsame experimentelle Modelle mit ähnlicher Größe des Gehirns führen. Allerdings gibt es keine veröffentlichten Arbeit, unser Wissen, dass eine umfassende Darstellung, wie die zur Durchführung dieser Immundetektion Verfahren im gesamten Affenhirn bietet. Was folgt, ist eine detaillierte Beschreibung, wie eine ganze Affenhirn für großflächige immunhistochemischen Nachweis von verschiedenen Zielproteine ​​vorzubereiten. Diese Arbeit wurde als Ergebnis der Zusammenarbeit zwischen kommerziellen und akademischen Bemühungen entstanden. Als solche Einzelheiten im Zusammenhang mit Gewebe Einbettung und Schneiden ein proprietäres Wissen NSA bleiben. Teil 1: Animal Behandlung und Gewebe Vorbereitung Das Gehirn eines Erwachsenen grünen Meerkatze (Cercopithecus aethiops) ist für das vorliegende Protokoll verwendet. Alle Verfahren sind in Übereinstimmung mit den Canadian Council on Animal Care (CCAC) Richtlinien für die Nutzung und Pflege von Tieren in der biomedizinischen Forschung 1 durchgeführt. <ol><li> Tiere ist tief mit Ketaminhydrochlorid (10 mg / kg, im), mit einer Überdosis Natrium-Pentobarbital (25 mg / kg, iv) und perfundierten transkardial mit 0,1 M PBS, bis sie vollständig ausgeblutet eingeschläfert sediert. </li><li> Dies wird durch eine 4% Paraformaldehyd-Lösung in PBS für 5 min (~ 1 Liter) folgten. </li><li> Die ausgelagerten Gehirn ist in abgestuften PBS-gepufferte Saccharose-Lösungen (10, 20 und 30%, in der Reihenfolge) mit 0,02% Natriumazid gelegt und bei 4 ˚ C, bis das Gehirn sinkt auf den Boden des Behälters. Die Lösung ersetzt wird alle paar Tage, bis Gehirn durchläuft Gefrier-Schnitte. </li><li> Zu ihren proprietären MultiBrain unterzogen werden ™ Einbettung und Frost-Schneiden-Technologie; Das Gehirn ist in den Neurowissenschaften Associates (Knoxville, TN NSA) gesendet. </li><li> Sieben (7) Ausrichtung Sehenswürdigkeiten sind in der Einbettungsmatrix platziert, so dass Abschnitte werden korrekt ausgerichtet (dh links-rechts-Orientierung) und neu ausgerichtet nach histologischen Verarbeitung abgeschlossen ist. </li><li> Der gefrorene Block wird digital auf den Block Gesicht fotografiert vor jedem seriellen Abschnitt aus dem Block gesammelt wird. Digitale Bilder sowie Teile von diesem Gehirn kann später zur 3D-Rekonstruktion der anatomischen und histologischen Karten verwendet werden, bzw.. </li><li> Serielle frei schwebenden koronalen Abschnitte, bei einer Dicke von 50 um pro Abschnitt werden aus dem gesamten Gehirn gesammelt. </li></ol> Teil 2: Histologische Bearbeitung Die Schnitte werden in einem bestimmten räumlichen Abstand gewählt (zB 500-um) und für unsere experimentelle Zwecke wurden mit den folgenden Antikörpern verarbeitet: fragile-X mentale Retardierung Protein (FMRP; Chemicon; Temecula, CA), SMI32 (Sternberger Monoklonale Antikörper Inc.; Baltimore, MD) und NeuN (Chemicon; Temecula, CA). FMRP ist ein zytoplasmatische Protein, das reichlich in den Nervenzellen von normalen und Permutation Träger Gehirne 2 gefunden. NeuN (Neuronal Nuclei) erkennt spezifisch die DNA-bindende Neuronen-spezifische Protein NeuN die in den meisten Neuronen ist und in neuronalen Kerne, Perikarya und einige proximalen neuronale Prozesse 3 verteilt. SMI-32 ist ein monoklonaler Antikörper, der die nicht-phosphorylierten Epitop auf Neurofilament Proteinen 4 erkennt. <ol><li> Im Abstand von 500 um, sind etwa 140 Teile pro Antikörper verwendet. </li><li> Alle Inkubationen, Waschungen und Färbungen Schritte werden mit leichtem Rühren mit spezialisierten Färbung Schüsseln und Körbe (; Knoxville, TN HistoTools) durchgeführt. </li><li> Die Schnitte werden zunächst in einer Lösung mit 0,1 M PBS/0.3% Triton X-100 (TX) / 5% normal heiser Serum (NHS) für 60 min, um unspezifische Bindung von Antikörper-Moleküle und die daraus resultierenden Hintergrundfärbung zu reduzieren inkubiert. </li><li> Abschnitte für FMRP Immunfärbung markiert durchlaufen einen zusätzlichen Schritt der Antigen Wiederherstellung mithilfe Antigen Demaskierung Solution (Vector Labs, Burlingame, CA) nach Anweisungen des Herstellers. </li><li> Abschnitte werden dann über Nacht inkubiert und an unterschiedlichen Tagen in den jeweiligen Antikörper-Lösungen, die 0,1 M PBS/0.3% TX / 3% NHS (mit einem Verdünnungsfaktor von 1 / 5, 000 für FMRP und SMI-32 und 1 / 2, 000 für NeuN Antikörper. </li><li> 1 /; Am nächsten Tag sind die Abschnitte für die drei 10-min Perioden in Waschlösung (0,1 M PBS/0.3% TX) durch Inkubation für 2 h bei Raumtemperatur in biotinylierten Anti-Maus-sekundären Antikörper in Pferd angehoben (Vector Labs gewaschen, gefolgt 1000 Verdünnung in 0,1 M PBS/0.3% TX / 3% NHS). </li><li> Nach einem weiteren Satz von 3 10-min wäscht, sind die Abschnitte in einer Lösung aus Avidin-Biotin-konjugierte Meerrettichperoxidase-Komplex (Vector Labs) für 1 h bei Raumtemperatur gegeben. </li><li> Endlich, nach einem anderen Satz von wäscht, sind die Abschnitte für 10 min auf eine Nickel verbesserte dia unterzogenminobenzidine (Ni-DAB) Reaktion, die ein dunkelblaues innerhalb immunreaktiven Neuronen Fleck produziert. </li><li> Nach Abschluss der histologischen Verfahren, sind Abschnitte auf Gelatine-beschichteten Glasträger montiert. </li><li> Alle Abschnitte sind über Nacht luftgetrocknet und mit einem Deckglas nach Standardverfahren. </li></ol> Teil 3: Repräsentative Ergebnisse: Diese Methode produziert eine komplette Expressionsprofil eines Zielproteins von Interesse im gesamten Affenhirn. Hier zeigen wir repräsentative koronalen Abschnitte, die eine Momentaufnahme der FMRP, NeuN und SMI32 Ausdruck in der gleichen Affenhirn bieten. <img src="/files/ftp_upload/1286/1286fig1.png" alt="" /> Abbildung 1: Die Färbung Schale (A) und Korb (B) für eine groß angelegte Batch-Verarbeitung der im Streubesitz befindlichen Abschnitte für Immundetektion verwendet. <img src="/files/ftp_upload/1286/1286fig2.png" alt="" /> Abbildung 2: Repräsentative koronalen Abschnitte aus einer grünen Meerkatze Gehirn für FMRP (A), NeuN (B) und SMI32 (C) Antikörper gefärbt.

<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.

Es gibt zwei kritische Schritte in diesem Verfahren, dass groß angelegte Nachweis von Proteinen in einer ganzen Affenhirn möglich zu machen. Eine davon ist die Einbettung und Schneiden-Protokoll, das die proprietäre Wissen NSA bleibt. Die andere ist die Verwendung der Färbung Schüsseln und Körbe von HistoTools zur Verfügung gestellt. Letzteres ermöglicht eine einfache und schnelle Handhabung von vielen (~ 40) Abschnitte zu einem bestimmten Zeitpunkt. Es bietet auch die Mittel für die gleichmäßige Behandlung a...

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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

Immunhistochemie (IHC) ist eine der am häufigsten verwendeten Labor-Techniken für die Detektion von Target-Proteinen<em&gt; In situ</em&gt;. Fragen über die Expressionsmuster eines Zielproteins über das gesamte Gehirn sind relativ leicht zu beantworten, wenn Sie IHC in kleinen Gehirne, wie die von Nagetieren. Doch die gleichen Fragen in großen und gewundenen Gehirne, wie die der Primaten stellt eine Reihe von Herausforderungen. Hier präsentieren wir einen systematischen Ansatz für die Immundetektion der Zielproteine ​​in einer erwachsenen Affen Gehirn. Dieser Ansatz stützt sich auf das Gewebe eingebettet und Schnitte Methodik der NeuroScience Associates (NSA) sowie Tools, die speziell für die Batch-Färbung der im Streubesitz befindlichen Abschnitten entwickelt. Es führt zu einer gleichmäßigen Färbung von einer Reihe von Abschnitten, die in einem bestimmten Intervall, stellt das gesamte Gehirn. Die daraus resultierenden gefärbten Schnitten kann auf eine Vielzahl von analytischen Verfahren unterzogen werden, um Protein-Spiegel, die Bevölkerung von Neuronen, die ein bestimmtes Protein zu messen.

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

Großflächige Immundetektion der Zielproteine ​​in der gesamten Primatengehirn wird durch den Einsatz von neuartigen Gewebe Einbettung und Schneiden Methoden mit dem Einsatz von kreativen Apparat für Batch-Färbung von mehreren frei schwebenden Abschnitte zu einem bestimmten Zeitpunkt in Kombination möglich.

Batch Immunfärbung für Large-Scale Protein-Nachweis im Whole Affenhirn

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

Research

JoVE Journal

Biology

12.6K Aufrufe.  Montreal Neurological Institute. Großflächige Immundetektion der Zielproteine ​​in der gesamten Primatengehirn wird durch den Einsatz von neuartigen Gewebe Einbettung und Schneiden Methoden mit dem Einsatz von kreativen Apparat für Batch-Färbung von mehreren frei schwebenden Abschnitte zu einem bestimmten Zeitpunkt in Kombination möglich.

Serie: 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.

Large-Scale-Bildschirme von Metagenombanken

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

Forschung

Biologie

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.

Detektion von Protein Ubiquitination

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

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.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

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.

Die Reinigung des M. magneticum Stamm AMB-1 Magnetosom assoziierten Protein 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 Zebrafisch-Based In-vivo- Small Molecule-Screen

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 für Groß SNP Genotypisierung Anwendungen

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.

Eine Strategie für einfühlsam, 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 ...

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.

Untersuchung von Protein-Protein-Interaktionen in lebenden Zellen Mit Biolumineszenz-Resonanz-Energie-Transfer

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: Eine groß angelegte Methode für die Mehrparameter-Quantifizierung des Hefewachstums

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.

Identifizierung neuartiger Regulatoren der Pflanzentranspiration durch großangelegtes Thermisches Bildgebungsscreening 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 ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

Ein halbautomatisches ChIP-Seq-Verfahren für groß angelegte epigenetische Studien

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...