Wir danken der USGS und die Vermont Water Resources and Lake Studies Center für die Bereitstellung von Mitteln.

1. Phosphorus Extraction <ol><li> Bereiten Sie eine 1L Lösung von 0,25 M Natronlauge (NaOH, 40,00 g / mol) + 0,05 M Ethylendiamintetraessigsäure (EDTA, 292,24 g / mol). </li><li> 30 ml der NaOH + EDTA-Lösung bis 2 g des nassen oder getrockneten Boden / Probe in einem 50 mL konischen Rohr. </li><li> Inkubieren unter Rühren für 16 h bei Raumtemperatur. </li><li> Centrifuge 3000 xg für 3 Minuten. </li></ol> 2. Probenextrakt pH-Regelung <ol><li> Übertragen einer 0,1 ml Aliquot auf einem 1,5 mL Mikro-Zentrifugenröhrchen. </li><li> Add 0.017 mL 2,5 M Essigsäure, 0,144 ml 0,4 M Acetatpuffer (70,5% von Natriumacetat [CH 3 COONa, 82,03 g / mol] und 29,5% Essigsäure, pH 5,0) und 0,739 ml deionisiertem Wasser. </li><li> Das Endvolumen eingestellt Extrakt wird 1 mL und der endgültige pH-Wert 5,0 liegen. </li></ol> 3. Enzyme Stammlösung Vorbereitung <ol><li> Bereiten Sie 1 L von 0,1 M Natriumacetat (CH 3 COONa, 82,03 g / mol)-Lösung, pH 5,0. </li><li> Bereiten Sie 2 x 10 ml saure Phosphatase Stammlösungen in der Natrium-Acetat-Puffer in Schritt 3.1, so dass Endkonzentrationen je 0,5 Einheiten / ml vorbereitet sind. Saure Phosphatase Typ I aus Triticum aestivum (Weizenkeime, GP, in der Regel 0,5 U mg-1 fest, EC 3.1.3.2) Saure Phosphatase Typ IV-S aus Solanum tuberosum (Kartoffel, PP, in der Regel 4,6 U mg-1 fest, EC 3.1.3.2) * Einheiten der Aktivität werden durch den Lieferanten, wobei 1 Unit als die Befreiung von 1 Mikromol Orthophosphat, um die Lösung pro Stunde bei 37 ° C definiert angegeben </li><li> Lyophilisierte Nuklease P1 (EG 3.1.30.1) aus Penicillium citrinum (Pilze, NP1, in der Regel 500 U mg -1 fest) in einem mL VE-Wasser-dieser kann nun bei 4 ° C gelagert werden für eine spätere Verwendung. </li><li> Pipette NP1 in einem der 10 ml-Röhrchen in Schritt 3.2, so dass die Endkonzentration 2,5 Einheiten / mL NP1 ist bereit, sanft durch mehrmaliges Wenden mischen. </li><li> Centrifuge die beiden 10 mL Enzymlösungen 3000 xg für 30 Minuten. </li></ol> 4. P Calibration Curve and Controls <ol><li> Bereiten Sie 1 l 1 mM Kaliumphosphat (K2HPO4, 174,18 g / mol) Stammlösung. </li><li> 1 ml der 1 mM Kaliumphosphat-Stammlösung in ein 1,5 ml Zentrifugenröhrchen und führen sieben 0,5 ml Verdünnungsreihen. </li><li> Entsorgen Sie die erste Röhre, so dass Sie 7 Verdünnungen von 20 nmol-0,625 nmol P. haben </li><li> Transfer-80 ul aus jedem Röhrchen in zweifacher Ausfertigung, um Zeilen B - H eines Standard-96-Well-Platte. </li><li> Add 80 ul von Natrium-Acetat-Puffer (Abschnitt 3.1) zu A der Spalten 11 und 12-dies ist die 0 nmol P Kalibrierpunkt. Reihe </li><li> Jede Spalte 11 und 12 nun enthält 8 Referenzproben von 0 nmol bis 20 nmol anorganischem Phosphat. </li><li> Spalte 10 enthält die folgenden Steuerelemente: <ol><li> Add 40 ul PP + GP Enzymlösung + 40 ul Natriumacetatpuffer (Abschnitt 3.1) zu den ersten drei Brunnen in Spalte 10. </li><li> Zugabe von 40 ul PP + GP + NP1 Enzymlösung + 40 ul Natriumacetatpuffer auf die nächsten drei Brunnen. </li><li> Zugabe von 40 ul Natriumacetatlösung mit 10 nmol Glucose-6-Phosphat (C 6 H 11 Na 2 O 9 P &amp;bullet; xH 2 O, 304,1 g / mol) + 40 ul PP + GP Enzymlösung auf eine gut und die endgültige gut in Spalte 10 add Natriumacetatlösung statt Enzymlösung. </li></ol></li></ol> 5. Sample + Enzyminkubation <ol><li> Jede Probe getestet wird belegen die ersten 9 Vertiefungen in 1 Reihe mit einem Standard 96-Well-Platte. </li><li> Verteilen Sie 40 ul pH-eingestellten Sample Auszüge aus Kapitel 2 in die Vertiefungen 1-9 in bis zu 8 Zeilen. </li><li> Nachdem alle Proben verteilt wurden * Verwendung einer Mehrkanal-Pipette PP + GP Enzymlösung Spalten 1-3, PP + GP + NP1 Spalten 4-6 und Natriumacetatpuffer (hergestellt in Abschnitt 3.1), Spalten 7-9. Verteilen * Dieser Schritt muss schnell durchgeführt werden, um sicherzustellen, alle Proben erhalten gleichwertige Inkubationszeit. </li><li> Decken Sie die 96-Well-Platte mit einem Deckel und inkubieren Proben + Enzymlösungen, Kontrollen und Kalibrierkurve genau 1 Stunde bei 37 ° C. </li></ol> 6. Kolorimetrische Messung der Released und Hintergrund Anorganische P <ol><li> Bereiten Sie 50 mL von jeder der folgenden Lösungen in VE-Wasser: Lösung A *: 0,1 M Ascorbinsäure (C6H8O6, 176,12 g / mol) + 0,5 M Trichloressigsäure (Cl3CCOOH, 163,39 g / mol) Lösung B: 0,01 M Ammoniummolybdat ((NH4) 2MoO4, 196,01 g / mol) Lösung C: 0,1 M Natriumcitrat (HOC (COONa) (CH2COONa) 2 2H2O, 294,10 g / mol) + 0,2 M Natriumarsenat (NaAsO2, 129,91 g / mol) + 5% Eisessig (CH3CO2H, 60,05 g / mol) * Lösung A muss täglich zubereitet werden. </li><li> Fügen Sie 25 ul Lösung SDS, 100 ul Lösung A, 20 ul Lösung B und 50 ul und der Lösung C in alle Vertiefungen in den 96-Loch-plaß. Tun Sie dies schnell mit einer Mehrkanal-Pipette. </li><li> Die Platte abdecken und inkubieren 30 Minuten bei Raumtemperatur. </li><li> Die Absorption bei 850 nm in einer abstimmbaren Mikro-Platten-Lesegerät. </li></ol> 7. Klassifikation der P-Verbindungen <ol><li> Import von Rohdaten in ein Tabellenkalkulationsprogramm (zB Microsoft Excel). </li><li> Plot der anorganischen Phosphor-Eichkurve: 0 bis 40 nmol P auf der x-Achse und den Mittelwert der doppelten Absorptionsmessungen auf der y-Achse. </li><li> Führen Sie eine lineare Regression und finden Sie die Gleichung der Trendlinie. </li><li> Spalten 1-3: Direkt bewerben die Eichkurve, das ist Hintergrund anorganischen P. </li><li> Spalten 4-6: Durchschnittliche dreifacher Ausfertigung Abtastwerte und subtrahieren Kontrollen (Enzymlösung + Hintergrund anorganischen P) von den Brutto-Absorption, das ist P aus hydrolysierbaren P Monoester abgeleitet. </li><li> Spalten 7-9: Gleiche wie 7,5 bis subtrahieren Absorptionswerte aus den Spalten 4-6 - das ist P aus hydrolysierbaren P Diester. </li><li> Convert Absorption Daten nmol P durch die Anwendung der Kalibrierung Gleichung freigegeben. </li><li> nmol P freigesetzt werden können zurück zu mg P / kg des ursprünglichen Bodens bezogen werden. Alternativ kann die Beurteilung Anteile der einzelnen P-Klasse auch ein nützlicher Ansatz für die Analyse von Daten. </li></ol> 8. Repräsentative Ergebnisse: Eine schnelle visuelle Überprüfung der 96-Well-Platte nach dem kolorimetrischen Chemie bietet Anhaltspunkte, ob das Verfahren korrekt durchgeführt wurde. Das erste, was zu überprüfen ist das Niveau der Flüssigkeit in jede Vertiefung durch das Scannen der Seite Profil der Platte. Es sollte genau 275 ul von Reagenzien in alle Vertiefungen werden. Als nächstes Sichtprüfung der Farbe des dreifachen Probenvertiefungen und doppelte Kalibrierung Brunnen. Diese technischen replizieren sollten die gleichen Blauton sein. Anschließend eine Eichkurve, die beiden Brunnen, die Glucose-6-Phosphat und überprüfen sie veröffentlicht alle 10 nmol von anorganischen P. Nach insgesamt extrahierten P gemessen wurde mittels ICP-OES oder eine alternative Methode, sicherzustellen, dass die Gesamt-P berechneten Werte mit dieser Protokoll nicht den Betrag übersteigen, von P, extrahiert wurde. Hinweis: Eine sorgfältige Datenverwaltung in der Tabelle wird der Hut vor quantitativen Fehlern helfen. Sie werden mit viel Zahlen zu tun auf einmal so eine Vorlage erstellen, wird die Zeit gut verbracht werden. <img src="/files/ftp_upload/2660/2660fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Ein 96-Well-Platte, welche die Ergebnisse für 8 Proben (Reihen AH) und eine Eichkurve (Spalten 11 und 12). Kontrollen sind in Spalte 10. Farbintensität zu erhöhen zwischen den Spalten 1-3 und 4-6 ist darauf zurückzuführen, hydrolysiert Monoester P-Verbindungen, zwischen den Spalten 4-6 und 7-9 ist darauf zurückzuführen, hydrolysiert Diester P-Verbindungen. <img src="/files/ftp_upload/2660/2660fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Verteilung der P-Klassen in einer 0,25 M NaOH-0.05 M EDTA-Extrakt eines Vermont Bodenprobe mit High-Throughput-enzymatische Hydrolyse. Fehlerbalken zeigen die Standardabweichung, 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>

Von Natur erfordert eine schnelle Methode mit kleinen Volumina großer Sorgfalt. Deshalb ist die wichtigste Schritte sind solche, die Pipettieren Lösungen auf den Teller. Präzise, ​​und vor allem konsistente Pipette Technik sind wesentlich für den Erfolg dieses Tests. Die NaOH-EDTA-Extraktion wird es den meisten P in vielen Proben in drei Klassen charakterisiert werden: Orthophosphat, Monoester und Diester P P. Böden, Dünger, Sedimente oder andere umweltschonende Probe, die NaOH-EDTA-extrahierbare P charakterisiert werden kann enthält . P bildet in Umweltproben sind nicht unbedingt stabil und diese Technik wird sichergestellt, Proben charakterisiert werden, bevor Proben, ohne dass ein Heer von Forschern beschäftigen, sind gefährdet. Dieser Test eignet sich besonders, wenn eine große Anzahl von Proben bearbeitet werden sollen. Das Reagenz muss und Platzbedarf wurden bis auf ein erträgliches Maß (zB 1,5 ml Mikrozentrifugenröhrchen anstatt 50 mL Glaskolben) skaliert. Diese Anpassung auch Grenzen von Abfällen.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </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> Eisessig </td><td> Sigma-Aldrich </td><td> 242853 </td></tr><tr><td> Natriumacetat </td><td> Sigma-Aldrich </td><td> S2889 </td></tr><tr><td> Weizen Acid Phosphatase </td><td> Sigma-Aldrich </td><td> P3627 </td></tr><tr><td> Potato Acid Phosphatase </td><td> Sigma-Aldrich </td><td> P1146 </td></tr><tr><td> Nuclease P1 </td><td> Sigma-Aldrich </td><td> N8630 </td></tr><tr><td> Kaliumphosphat </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-Phosphat </td><td> Sigma-Aldrich </td><td> G7250 </td></tr><tr><td> SDS </td><td> Sigma-Aldrich </td><td> L4390 </td></tr><tr><td> Askorbinsäure </td><td> Sigma-Aldrich </td><td> A5960 </td></tr><tr><td> TCA </td><td> Sigma-Aldrich </td><td> T9159 </td></tr><tr><td> Ammoniummolybdat </td><td> Sigma-Aldrich </td><td> A1343 </td></tr><tr><td> Natriumzitrat </td><td> Sigma-Aldrich </td><td> S1804 </td></tr><tr><td> Natriumarsenat </td><td> Sigma-Aldrich </td><td> S9663 </td></tr></tbody></table>

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

Viele Arten von organischen Phosphor (P)-Moleküle existieren in Umweltproben 1. Traditionelle P Messungen nicht erkennen diese organischen P-Verbindungen, da sie nicht mit kolorimetrischen Reagenzien 2,3 reagieren. Die enzymatische Hydrolyse (EH) ist ein aufstrebendes Verfahren zur genauen Charakterisierung organischer P bildet in Umweltproben 4,5. Diese Methode ist nur in der Genauigkeit von Phosphor-31 Nuclear Magnetic Resonance Spectroscopy (31 P-NMR)-eine Methode, die teuer ist und erfordert spezielle technische training6 übertrumpft. Wir haben eine enzymatische Hydrolyse-Methode für die Messung von drei Klassen von Phosphor (P Monoester, Diester P und anorganischen P) zu einem Mikroplatten-Reader System 7 angepasst. Diese Methode bietet den Forschern eine schnelle, präzise, ​​kostengünstig und anwenderfreundlich bedeutet P Spezies in Böden, Sedimenten, Dünger und wenn konzentriert, aquatischen Proben zu messen. Dies ist die einzige High-Throughput-Verfahren zur Messung der Formen und Enzym-Labilität von organischen P, die in einem Standard-Labor durchgeführt werden kann. Die resultierenden Daten einen Einblick für Wissenschaftler, System Nährstoffgehalt und Eutrophierungspotenzial.

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

Dieses Protokoll beschreibt eine High-Throughput-Verfahren zur enzymatischen Hydrolyse, dass ein Mikroplatten-Reader nutzt, um zu messen und zu klassifizieren Boden Phosphor als P Mono-, Di-P und anorganischen P. Bis zu 96 Proben auf einmal in einem Standard-Labor gemessen werden.

High-Throughput Measurement and Classification of Organic P in Umweltproben

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 Aufrufe.  University of Vermont. Dieses Protokoll beschreibt eine High-Throughput-Verfahren zur enzymatischen Hydrolyse, dass ein Mikroplatten-Reader nutzt, um zu messen und zu klassifizieren Boden Phosphor als P Mono-, Di-P und anorganischen P. Bis zu 96 Proben auf einmal in einem Standard-Labor gemessen werden.

Serie: 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.

High-Throughput-Screening und Biosensorik mit Fluorescent C. elegans Die Stämme

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

Forschung

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.

High Throughput Screening von Fungal Endoglucanaseaktivität in 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 Quantenausbeute Messung von Pulverproben

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.

Chemisch-Antikörper blockiert Microarray für Multiplex-High-Throughput-Profiling von spezifischen Protein-Glykosylierung bei 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 ...

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.

Ein High-Throughput-Verfahren zur Messung der glomerulären Filtrationsrate in Conscious Mäuse

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.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

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.

Präzise, Hochdurchsatz-Analyse des Bakterienwachstums

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.

Hochdurchsatz-Messung von Dictyostelium Discoideum Macropinocytosis von Flow Cytometry

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.

Hochdurchsatz-Messung der Darm Transitzeit mit Larven Zebrafisch

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;