Nous remercions l&#39;USGS et le Vermont, Ressources en eau et d&#39;études Lac Centre de fournir un financement.

1. Extraction du phosphore <ol><li> Préparer une solution 1L de 0,25 M d&#39;hydroxyde de sodium (NaOH, 40,00 g / mol) + 0,05 M acide éthylènediaminetétraacétique (EDTA, 292,24 g / mol). </li><li> Ajouter 30 ml de la solution de NaOH + EDTA à 2 g de sol humide ou sec / échantillon dans un tube conique de 50 ml. </li><li> Incuber sous agitation pendant 16 h à température ambiante. </li><li> Centrifugeuse 3000 xg pendant 3 minutes. </li></ol> 2. Ajustement du pH de l&#39;échantillon Extrait <ol><li> Transférer une partie aliquote de 0,1 mL à 1,5 mL une micro-centrifugeuse tube. </li><li> Ajouter 0,017 ml de 2,5 M d&#39;acide acétique, 0,144 ml de tampon acétate 0,4 M (70,5% d&#39;acétate de sodium [CH 3 COONa, 82,03 g / mol] et 29,5% d&#39;acide acétique, pH 5,0), et 0,739 ml d&#39;eau déminéralisée. </li><li> Le volume final de l&#39;extrait sera ajusté de 1 mL et le pH final sera 5,0. </li></ol> 3. Préparation de la solution enzymatique Stock <ol><li> Préparer 1 L d&#39;acétate de sodium 0,1 M (CH 3 COONa, 82,03 g / mol) en solution, pH 5,0. </li><li> Préparer 2 x 10 ml des solutions d&#39;acide phosphatase stock dans le tampon acétate de sodium préparée à l&#39;étape 3.1 de telle sorte que les concentrations finales sont chacun 0,5 unités / ml. Phosphatase acide de type I de Triticum aestivum (blé germe; GP, généralement de 0,5 mg U-1 solide, EC 3.1.3.2) Phosphatase acide de type IV-S de Solanum tuberosum (pomme de terre; PP, généralement 4,6 mg U-1 solide, EC 3.1.3.2) * Les unités d&#39;activité sont spécifiés par le fournisseur lorsque une unité est définie comme la libération de 1 micromole de l&#39;orthophosphate de la solution par heure à 37 ° C. </li><li> Reconstituer lyophilisé nucléase P1 (EC 3.1.30.1) de Penicillium citrinum (champignons; NP1, généralement 500 mg U -1 solide) dans un ml de l&#39;eau déminéralisée, ce qui peut désormais être conservés à 4 ° C pour une utilisation future. </li><li> Pipeter NP1 dans l&#39;un des tubes de 10 ml préparée à l&#39;étape 3.2 telles que la concentration finale est de 2,5 unités / ml NP1; mélanger délicatement en retournant plusieurs fois. </li><li> Centrifuger les deux 10 ml solutions enzymatiques 3000 xg pendant 30 minutes. </li></ol> 4. Courbe d&#39;étalonnage P et contrôles <ol><li> Préparer 1 L de phosphate de potassium 1 mM (K2HPO4, 174,18 g / mol) solution stock. </li><li> Ajouter 1 mL de la 1 mM de phosphate de potassium de la solution mère dans un tube à centrifuger de 1,5 ml et 0,5 ml accomplisse sept dilutions successives. </li><li> Jeter le tube d&#39;abord si vous disposez de 7 dilutions allant de 20 nmol-0.625 nmol P. </li><li> Transfert 80 ul de chaque tube en double exemplaire aux lignes B - H d&#39;une plaque standard 96 puits. </li><li> Ajouter 80 ul de tampon acétate de sodium (point 3.1) à la ligne A de colonnes 11 et 12, tel est le point 0 nmol calibration P. </li><li> Chaque colonne 11 et 12 contient maintenant 8 échantillons de référence de 0 à 20 nmol phosphate inorganique nmol. </li><li> Colonne 10 contient les contrôles suivants: <ol><li> Ajouter 40μL PP + solution enzymatique GP + 40 ul de tampon acétate de sodium (section 3.1) pour les trois premiers puits dans la colonne 10. </li><li> Ajouter 40 uL PP + GP + solution enzymatique NP1 + 40 ul de tampon acétate de sodium pour les trois prochaines puits. </li><li> Ajouter 40 uL solution d&#39;acétate de sodium contenant 10 glucose-6 phosphate nmol (C 6 H 11 Na 2 O 9 P &amp;bullet; xH 2 O, 304,1 g / mol) + 40 uL PP + solution enzymatique GP d&#39;un puits et au bien-finales dans la colonne 10 une solution d&#39;acétate de sodium au lieu d&#39;ajouter la solution d&#39;enzyme. </li></ol></li></ol> 5. Échantillon + enzyme incubation <ol><li> Chaque échantillon testé occuperont les 9 premiers puits dans une rangée d&#39;une plaque standard 96 puits. </li><li> Distribuer 40 ul d&#39;extraits d&#39;échantillons à pH ajusté à la section 2 aux puits 1-9 sur jusqu&#39;à 8 lignes. </li><li> Après tous les échantillons ont été distribués * utiliser une pipette multicanaux pour distribuer PP + solution enzymatique GP à colonnes 1-3, PP + GP + NP1 aux colonnes 4-6 et un tampon d&#39;acétate de sodium (préparée à la section 3.1) dans les colonnes 7-9. * Cette étape doit être effectuée rapidement afin d&#39;assurer tous les échantillons obtenir du temps d&#39;incubation équivalent. </li><li> Recouvrir la plaque de 96 puits avec un couvercle et incuber des échantillons + solutions enzymatiques, les contrôles et courbe d&#39;étalonnage exactement 1 heure à 37 ° C. </li></ol> 6. Mesure colorimétrique de Sortie et P Contexte inorganiques <ol><li> Préparer 50 ml de chacune des solutions suivantes dans l&#39;eau déminéralisée: Solution A *: 0,1 M d&#39;acide ascorbique (C6H8O6, 176,12 g / mol) + 0,5 M d&#39;acide trichloracétique (CI3CCOOH, 163,39 g / mol) Solution B: 0,01 M molybdate d&#39;ammonium ((NH4) 2MoO4, 196,01 g / mol) Solution C: 0,1 M citrate de sodium (HOC (COONa) (CH2COONa) 2 2H2O, 294,10 g / mol) + 0,2 M arséniate de sodium (NaAsO2, 129,91 g / mol) + 5% d&#39;acide acétique glacial (CH3CO2H, 60,05 g / mol) * Solution A doit être préparée chaque jour. </li><li> Ajouter 25 ul d&#39;une solution de SDS, 100 pi de solution A, 20 ul de la solution B et 50 pi et de la solution C à tous les puits dans la pl de 96 puitsmangé. Pour ce faire, rapidement avec une pipette multicanaux. </li><li> Couvrir la plaque et laisser incuber 30 minutes à température ambiante. </li><li> Mesurer l&#39;absorbance à 850 nm dans toute accordable micro-lecteur de plaques. </li></ol> 7. Classification des composés P <ol><li> Importer des données brutes dans un tableur (par exemple Microsoft Excel). </li><li> Tracer la courbe d&#39;étalonnage de phosphore inorganique: 0 à 40 nmol P sur l&#39;axe des x et la moyenne des mesures d&#39;absorbance en double sur l&#39;axe des y. </li><li> Effectuer une régression linéaire et de trouver l&#39;équation de la droite de meilleur ajustement. </li><li> Colonnes 1-3: appliquer directement la courbe d&#39;étalonnage-ce sont les antécédents inorganiques P. </li><li> Colonnes 4-6: Les valeurs moyennes de l&#39;échantillon en triple et soustraire des contrôles (solution enzymatique + P inorganique de fond) de l&#39;absorbance brute-ce n&#39;est dérivée de P P monoesters hydrolysables. </li><li> Colonnes 7-9: Même chose que 7,5 exception des valeurs d&#39;absorbance soustraire colonnes 4-6 - c&#39;est à partir de diesters P P hydrolysables. </li><li> Convertir les données d&#39;absorbance à nmol P libéré en appliquant l&#39;équation d&#39;étalonnage. </li><li> nmol P libérée peut être liée au retour mg P / kg de sol d&#39;origine. Sinon, l&#39;évaluation des proportions de chaque classe P peut aussi être une approche utile pour l&#39;analyse des données. </li></ol> 8. Les résultats représentatifs: Une inspection visuelle rapide de la plaque de 96 puits, après la chimie colorimétrique offrira des indices pour savoir si la procédure a été effectuée correctement. La première chose à vérifier est le niveau de liquide dans chaque puits en scannant le profil latéral de la plaque. Il doit être exactement 275 uL de réactifs dans tous les puits. Ensuite, inspecter visuellement la couleur du puits d&#39;échantillon triple et double puits d&#39;étalonnage. Ces techniques devraient être de reproduire la même nuance de bleu. Ensuite appliquer la courbe de calibration pour les deux puits contenant du glucose-6-phosphate et de vérifier, ils sortent tous les 10 nmol de P. Après inorganiques P extrait total a été mesurée en utilisant l&#39;ICP-OES ou une autre méthode, assurez-vous que le P total des valeurs calculées à l&#39;aide de cette protocole de ne pas dépasser la quantité de P qui a été extrait. Remarque: Attention gestion des données dans le tableur de se prémunir contre des erreurs quantitatives. Vous devrez traiter avec beaucoup de numéros à la fois, afin de créer un modèle sera du temps bien investi. <img src="/files/ftp_upload/2660/2660fig1.jpg" alt="Figure 1" /> Figure 1. Une plaque de 96 puits montrant les résultats de 8 échantillons (lignes AH) et une courbe de calibration (colonnes 11 et 12). Les contrôles sont dans la colonne 10. Augmentation de l&#39;intensité des couleurs entre les colonnes 1-3 et 4-6 est due à des composés hydrolysés monoester P, entre les colonnes 4-6 et 7-9 est due à des composés hydrolysés P diester. <img src="/files/ftp_upload/2660/2660fig2.jpg" alt="Figure 2" /> Figure 2. Répartition des classes de P dans un 0,25 M de NaOH 0,05 M d&#39;EDTA extraction d&#39;un échantillon de sol à l&#39;aide du Vermont à haut débit hydrolyse enzymatique. Les barres d&#39;erreur indiquent l&#39;écart type, n = 3.

<ol>
	<li>Turner, B. L., Frossard, E., Baldwin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organic phosphorus in the environment. , (2005).</li><li>Murphy, J., Riley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+single+solution+method+for+determination+of+phosphate+in+natural+waters.">A modified single solution method for determination of phosphate in natural waters.</a> Anal. Chim. Acta. 27, 31-36 (1962).</li><li>Dick, W. A., Tabatabai, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+orthophosphate+in+aqueous+solutions+containing+labile+organic+and+inorganic+phosphorus+compounds.">Determination of orthophosphate in aqueous solutions containing labile organic and inorganic phosphorus compounds.</a> J. Environ. Qual. 6, 82-85 (1977).</li><li>He, Z., Toor, G. S., Honeycutt, C. W., Sims, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+hydrolysis+approach+for+characterizing+labile+phosphorus+forms+in+dairy+manure+under+mild+assay+conditions.">An enzymatic hydrolysis approach for characterizing labile phosphorus forms in dairy manure under mild assay conditions.</a> Bioresour. Technol. 97, 1660-1668 (2006).</li><li>He, Z., Honeycutt, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+molybdenum+blue+method+for+orthophosphate+determination+suitable+for+investigating+enzymatic+hydrolysis+of+organic+phosphates.">A modified molybdenum blue method for orthophosphate determination suitable for investigating enzymatic hydrolysis of organic phosphates.</a> Commun. Soil Sci. Plant Anal. 36, 1373-1383 (2005).</li><li>Cade-Menun, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+phosphorus+in+environmental+and+agricultural+samples+by+31P+nuclear+magnetic+resonance+spectroscopy.">Characterizing phosphorus in environmental and agricultural samples by 31P nuclear magnetic resonance spectroscopy.</a> Talanta. 66, 359-371 (2005).</li><li>Johnson, N. R., Hill, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+species+composition+of+poultry+manure-amended+soil+using+high-throughput+enzymatic+hydrolysis.">Phosphorus species composition of poultry manure-amended soil using high-throughput enzymatic hydrolysis.</a> Soil Sci. Soc. Am. J. 74, 1786-1791 (2010).</li></ol>

Par sa nature même, une méthode rapide utilisant de petits volumes requiert le plus grand soin. Par conséquent, les étapes les plus critiques sont ceux qui impliquent des solutions de pipetage sur la plaque. Précis, et surtout, la technique de la pipette cohérents sont essentiels pour la réussite de ce test. L&#39;extraction NaOH-EDTA permettra la plupart du P dans de nombreux échantillons à être caractérisé en trois classes: orthophosphate, monoester et de diester P P. Sols, les engrais, les sédiments ou tout autre échantillon de l&#39;environnement qui contient du NaOH-EDTA-extractibles P peut être caractérisée . Formes de P dans les échantillons environnementaux ne sont pas forcément stables et cette technique va s&#39;assurer que les échantillons sont caractérisés avant que les échantillons sont compromis sans avoir besoin d&#39;employer une armée de chercheurs. Ce test est particulièrement adapté quand un grand nombre d&#39;échantillons doivent être traitées. Les besoins de réactifs et d&#39;espace ont été revues à la baisse à un niveau gérable (par exemple 1,5 ml microtubes plutôt que 50 flacons en verre ml). Cette adaptation limite également la production de déchets.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> NaOH </td><td> Sigma-Aldrich </td><td> S8045 </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> EDS </td></tr><tr><td> Acide acétique glacial </td><td> Sigma-Aldrich </td><td> 242853 </td></tr><tr><td> Acétate de sodium </td><td> Sigma-Aldrich </td><td> S2889 </td></tr><tr><td> Phosphatase acide blé </td><td> Sigma-Aldrich </td><td> P3627 </td></tr><tr><td> Phosphatase acide de pommes de terre </td><td> Sigma-Aldrich </td><td> P1146 </td></tr><tr><td> Nucléase P1 </td><td> Sigma-Aldrich </td><td> N8630 </td></tr><tr><td> Phosphate de Potassium </td><td> Sigma-Aldrich </td><td> P2222 </td></tr><tr><td></td><td> Sigma-Aldrich </td><td></td></tr><tr><td></td><td> Sigma-Aldrich </td><td></td></tr><tr><td> Glucose-6-phosphate </td><td> Sigma-Aldrich </td><td> G7250 </td></tr><tr><td> SDS </td><td> Sigma-Aldrich </td><td> L4390 </td></tr><tr><td> Acide ascorbique </td><td> Sigma-Aldrich </td><td> A5960 </td></tr><tr><td> TCA </td><td> Sigma-Aldrich </td><td> T9159 </td></tr><tr><td> Molybdate d&#39;ammonium </td><td> Sigma-Aldrich </td><td> A1343 </td></tr><tr><td> Citrate de sodium </td><td> Sigma-Aldrich </td><td> S1804 </td></tr><tr><td> Arséniate de sodium </td><td> Sigma-Aldrich </td><td> S9663 </td></tr></tbody></table>

high-throughput measurement and classification of organic p in environmental samples

De nombreux types de composés organiques du phosphore (P) de molécules existent dans des échantillons de l&#39;environnement 1. Traditionnelle mesures P ne détecte pas ces composés organiques P, car ils ne réagissent pas avec les réactifs colorimétriques 2,3. L&#39;hydrolyse enzymatique (EH) est une nouvelle méthode pour caractériser avec précision les formes organiques P dans les échantillons environnementaux 4,5. Cette méthode est seulement dans la précision forgées par le phosphore-31 spectroscopie par résonance magnétique nucléaire (31 P-RMN), une méthode qui est coûteux et nécessite spécialisés training6 technique. Nous avons adapté une méthode de l&#39;hydrolyse enzymatique capable de mesurer des trois classes de phosphore (P monoester, diester et P P inorganique) à un système de lecteur de microplaques 7. Cette méthode fournit aux chercheurs un moyen rapide, précis, abordable et convivial pour mesurer les espèces P dans les sols, les sédiments, les engrais et, si elle est concentrée, les échantillons aquatiques. C&#39;est la seule méthode à haut débit permettant de mesurer les formes et les enzymes labilité du P organique qui peut être effectuée dans un laboratoire standard. Les données résultant donne un aperçu de scientifiques qui étudient le contenu du système d&#39;éléments nutritifs et le potentiel d&#39;eutrophisation.

Watch this Scientific Journal Video about High-Throughput Measurement and Classification of Organic P in Environmental Samples at JoVE.com

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Ce protocole décrit une méthode à haut débit de l&#39;hydrolyse enzymatique qui utilise un lecteur de microplaques pour mesurer et classifier phosphore dans le sol que les monoesters P, P diesters et inorganiques P. Jusqu&#39;à 96 échantillons peuvent être mesurés en une seule fois dans un laboratoire standard.

High-Throughput mesure et de classification de P organique dans les échantillons environnementaux

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

high-throughput-measurement-classification-organic-p-environmental

Research

JoVE Journal

Biology

12.5K vues.  University of Vermont. Ce protocole décrit une méthode à haut débit de l'hydrolyse enzymatique qui utilise un lecteur de microplaques pour mesurer et classifier phosphore dans le sol que les monoesters P, P diesters et inorganiques P. Jusqu'à 96 échantillons peuvent être mesurés en une seule fois dans un laboratoire standard.

Vidéo: High-Throughput Measurement and Classification of Organic P in Environmental Samples

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Criblage à haut débit et de biodétection avec fluorescent C. elegans Souches

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Recherche

Biologie

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Criblage à haut débit activité endoglucanase fongique dans les Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absolute mesure du rendement quantique des échantillons de poudre

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Microarray Anticorps chimiquement bloqué pour multiplexée à haut débit de profilage de glycosylation des protéines spécifiques dans les échantillons complexes

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Une méthode à haut débit pour la mesure de débit de filtration glomérulaire chez des souris conscientes

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Analyse précise, haut débit de la croissance bactérienne

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Haut débit mesure de Dictyostelium discoideum macropinocytose par cytométrie en flux

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

Haut débit mesure du temps de Transit intestinale à l’aide de larve Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;