Ce travail a été soutenu par la NSF # 0920274 prix et de la Caroline du Sud Experiment Station Project (SC-1700340) pour KSS. Ce document est n ° Contribution Technique 5929 de la Station de Clemson University Experiment.

Le schéma d&#39;ensemble de ce protocole est présenté dans la figure 1. 1. Préparation de la solution pour les courbes d&#39;étalonnage et dosages <ol><li> Préparer 100 ml d&#39;une 2 mol / L solution d&#39;hydroxylamine-HCl. Peser 13,9 g de chlorhydrate d&#39;hydroxylamine (MW 69.49 g / mol) et dissoudre dans environ 50 ml d&#39;eau distillée-eau déminéralisée (FD 2 O). Ajuster le pH à 7,0 à l&#39;aide des pastilles d&#39;hydroxyde de potassium ou une solution concentrée. Amener le volume final de 100 ml. La solution peut être conservée à température ambiante pendant jusqu&#39;à 30 jours ou moins 4 ° C pendant jusqu&#39;à 90 jours. </li><li> Préparer 100 ml de chlorure ferrique / solution d&#39;acide chlorhydrique. Peser 13,5 g de chlorure ferrique (MW 270.32 g / mol) et dissoudre dans environ 50 ml ddH 2 O. Ajouter 41,3 ml d&#39;acide chlorhydrique concentré (12,1 mol / L) et porter à un volume final de 100 ml. La concentration finale de chlorure ferrique sera de 0,5 mol / L et la concentration finale d&#39;acide chlorhydrique sera de 5 mol/ L. La solution est stockée à température ambiante. </li><li> Préparer 100 ml d&#39;un 1,84 mol / L de solution d&#39;acide trichoroacetic. Peser 30,0 g d&#39;acide trichloracétique (MW 163.39 g / mol), dissoudre dans le trou DDH 2 O et porter le volume final de 100 ml. La solution est stockée à température ambiante. </li><li> Préparer 5 ml d&#39;une 0,1 mol / L solution de phosphate acétyle. Peser 0,092 g de phosphate acétyle (MW 184.06 g / mol), dissoudre dans le trou DDH 2 O et porter le volume final de 5 ml. Aliquoter la solution à microtubes (0,25 ml par tube) et congeler à -20 ° C jusqu&#39;à utilisation. Solution décongelés doivent être placés sur la glace et peut être utilisé pendant environ 1 heure. Solution de phosphate acétyle qui a été décongelé ne doit pas être recongelé et réutilisés. </li><li> Préparer 5 ml d&#39;une 0,1 mol / L de solution d&#39;ADP. Peser 0,24 g d&#39;ADP (MW 472.5 g / mol), dissoudre dans le trou DDH 2 O, et porter le volume final de 5 ml. Aliquoter la solution dans des microtubes (0,5 ml par tube) et congeler à -20 ° C jusqu&#39;àutilisation. Conserver la solution décongelée sur la glace jusqu&#39;à utilisation. </li><li> Préparer 50 ml d&#39;une 1 mol / L solution de Tris. Peser 6,06 g de Tris (MW 121.14 g / mol), et dissoudre dans 40 ml ddH 2 O. Ajuster le pH de la solution à 7,0 à l&#39;aide d&#39;acide chlorhydrique et porter le volume final de 50 ml. Conserver la solution à température ambiante. </li><li> Préparer 10 ml d&#39;une 1 mol / L de solution de chlorure de magnésium. Peser 2,03 g de chlorure de magnésium (MW 203.31 g / mol), dissoudre dans le trou DDH 2 O, et amener le volume final à 10 ml. Conserver la solution à température ambiante. </li><li> Préparer 25 ml solution de développement en mélangeant des volumes égaux de la solution acide de chlorure / ferrique chlorhydrique et la solution d&#39;acide trichloracétique. Solution de développement doit être préparé chaque jour et stockés à température ambiante. </li></ol> 2. Préparation d&#39;un courbe standard de phosphate acétyle <ol><li> Une courbe de phosphate acétyl standard est généré en utilisant des quantités connues de phosphate acétyle. Une courbe standard de six à eighpoints de t est approprié. Les normes devraient varier de 0,03 à 0,9 micromoles micromoles pour 300 uL, ce qui correspond à des concentrations finales de 0,1 à 3 mmol / L. Diluer la solution de phosphate acétyl achat d&#39;actions à des concentrations appropriées dans 100 mM Tris solution dans un volume final de 300 ul. Un échantillon témoin sans phosphate acétyl devrait également être préparée. </li><li> Incuber chaque norme et le contrôle à 37 ° C dans un bloc chauffant pendant 1 minute. </li><li> Ajouter 50 uL de la solution de chlorhydrate d&#39;hydroxylamine à chaque norme et le contrôle et mélanger en secouant trois fois. Placez la norme dans un bloc de 60 ° C chaleur et incuber pendant cinq minutes. </li><li> Ajouter 100 pi de solution de développement aux normes et le contrôle, mélanger en secouant trois fois, et permettre de développer de couleur d&#39;au moins une minute à température ambiante. </li><li> Centrifugeuse à toutes les normes et le contrôle dans une microcentrifugeuse à 18.000 xg pendant 1 minute. </li><li> Mesurer les échantillons par spectrophotométrie unet 540 nm en utilisant 1ml cuvettes en plastique, en utilisant la commande comme le blanc pour la spectrophotométrie. </li><li> Générer une courbe standard en utilisant une analyse appropriée des données et des logiciels graphiques (par exemple Kaleidagraph Synergie de logiciels). Valeurs de R 2 de 0,99 ou plus sont souhaitables. Les données sont tracées comme l&#39;absorbance à 540 nm par rapport pmoles de phosphate présents acétyle. </li></ol> 3. Dosage de l&#39;activité kinase Acétate <ol><li> En utilisant les solutions décrites, préparer un mélange réactionnel contenant une concentration finale de 0,1 tampon mol / L Tris, 10 mmol / L de chlorure de magnésium, 5 mmol / L d&#39;ADP, et 2 mmol / L de phosphate acétyle. Un mélange de 10 ml de réaction, il faudra 8,2 ml ddH 2 O, 1,0 ml de Tris solution, 0,1 ml de solution de chlorure de magnésium, 0,5 mL d&#39;ADP solution, et 0,2 ml solution de phosphate acétyle. Distribuer le mélange réactionnel dans 300 aliquotes de microtubes. Pour la détermination des paramètres cinétiques et optimisation des concentrations de substrat, un substrat peut êtrevariées, et l&#39;autre a tenu à une concentration constante dans les réactions. </li><li> Pré-incuber les réactions pendant 1 minute à 37 ° C dans un bloc thermique. </li><li> Ajouter la quantité désirée de l&#39;enzyme à la réaction et de retourner immédiatement le tube dans le bloc 37 ° C chaleur. Inclure également une réaction de contrôle à laquelle aucune enzyme est ajoutée. Si plusieurs réactions sont effectuées, ajouter enzyme pour les tubes à intervalles de temps spécifiques. Soyez sûr de garder trace du temps. (REMARQUE: La quantité d&#39;enzyme pour être utilisé devra être optimisé afin de garantir que tous les phosphates acétyle ou substrat ADP ne sera pas épuisé - voir la section Discussion) </li><li> Après 5 minutes, ajouter 50 pl de la solution de chlorhydrate d&#39;hydroxylamine à chaque réaction et le contrôle, mélanger, et placer les tubes dans un bloc de 60 ° C chaleur. Si plusieurs réactions sont effectuées, ce qui devrait être fait au même intervalle de temps que l&#39;enzyme a été ajouté. </li><li> Incuber les réactions et le contrôle pendant 5 minutes à 60 ° C. </li><li> Ajouter 100 &amp;mu; L de la solution de développement à chaque réaction et le contrôle, mélanger, et placer dans un rack tube sur la paillasse. Autoriser coloration se développer pendant au moins 1 minute. </li><li> Centrifuger tous les tubes dans une microcentrifugeuse à 18.000 xg pendant 1 minute. </li><li> Mesure des échantillons par spectrophotométrie à 540 nm en utilisant 1ml cuvettes en plastique et de déterminer la quantité de phosphates présents acétyl par comparaison à la courbe standard de phosphate acétyle. </li><li> La quantité de substrat de phosphate acétyl appauvri peut être déterminé en soustrayant la quantité de phosphate acétyl restant après la réaction enzymatique à partir de la quantité de phosphate présent dans l&#39;acétyl l&#39;enzyme de contrôle manquent. Effectuer l&#39;analyse de données en utilisant les données et les logiciels appropriés graphique (par exemple Kaleidagraph Synergie de logiciels). R 2 des valeurs supérieures à 0,97 sont souhaitables. </li></ol> 4. Les résultats représentatifs: Le but de ce test est de mesurer l&#39;activité kinase de l&#39;acétate dans la directeion de la formation d&#39;acétate. Ceci est fait en mesurant la consommation du substrat de phosphate acétyle, car il n&#39;ya pas facile, la mesure directe de la production d&#39;acétate. Phosphate de Acétyl restant après un temps donné lors de la réaction enzymatique est converti en hydroxamate acétyl et par la suite au complexe hydroxamate ferrique, qui a une couleur rougeâtre (figure 2) qui peut être mesurée à 540 nm. La courbe standard comploter absorbance en fonction actuelle micromoles de phosphate acétyle dans les normes est utilisé pour déterminer la quantité de phosphate acétyl restant dans chaque réaction. La courbe standard doit être linéaire par le point zéro. Une courbe représentant la norme montre la figure 3 a une pente de 1,2332 unités d&#39;absorbance par du phosphate présents pmole acétyle et une valeur R2 de 0,99, indiquant les données de l&#39;équation linéaire s&#39;adapte bien. L&#39;équation de la forme linéaire à la courbe standard de données est utilisée pour calculer la quantité de phosphate acétyl restant dans la réaction. Le micromolesde phosphate acétyl consommée est déterminée comme étant la différence entre les micromoles de phosphate acétyl présente dans la réaction de contrôle manquent d&#39;enzymes et de l&#39;acétyl micromoles de phosphate restant après la réaction enzymatique. Purifiée, recombinante M. acétate kinase thermophila a été dosé en utilisant le protocole décrit. Pour l&#39;expérience le montre la figure 4, la concentration d&#39;ADP a été maintenu constant à 5 mmol / L et la concentration de phosphate acétyle dans la réaction a été variée pour déterminer le K m pour le phosphate acétyle. Comme prévu, cette expérience démontre que l&#39;enzyme suit la norme de Michaelis-Menten-comme la cinétique pour chaque substrat et produit une courbe de saturation hyperbolique lors du traçage d&#39;activité versus concentration de phosphate acétyle. L&#39;apparente K m de valeur pour le phosphate était de 0,27 acétyl ± 0,01 mmol / L, ce qui se compare favorablement à la valeur publiée K m de 0,47 ± 0,01 mmol / L utilisant le couplage dedisent 13. Le dosage direct peut également être utilisé pour mesurer l&#39;activité des kinases acétate qui utilisent des substrats autres que l&#39;ATP, ce qui n&#39;est pas possible en utilisant le test standard couplé. Recombinant purifié E. acétate kinase 16 histolytica, qui produit PP i plutôt que l&#39;ATP dans le sens de l&#39;acétate de formage, a été soumis à ce test en utilisant le phosphate de sodium à la place de l&#39;ADP. La concentration de phosphate de sodium dans la réaction a été maintenu constant à 0,2 mol / L et la concentration en phosphate acétyl était varié. Quant à la M. acétate kinase thermophila, le E. acétate kinase histolytica suivi de Michaelis-Menten-comme la cinétique de phosphate acétyle et une courbe de saturation hyperbolique a été observée (figure 5). L&#39;apparente K m de valeur pour le phosphate était de 0,50 acétyl ± 0,006 mmol / L. Reeves et Guthrie 15 a déterminé une valeur de K m de 0,06 mmol / L pour le phosphate d&#39;acétyle pour l&#39;enzyme native, comment<html> Cependant, leur enzyme a été que partiellement purifiés et leur dosage impliqué quatre enzymes de couplage qui ont également été partiellement purifiés. Ainsi, il est difficile de comparer directement ces valeurs. Le schéma de protocole <ol><li> Générer une courbe standard de phosphate de l&#39;acétyl </li><li> Diluer l&#39;enzyme à la concentration désirée </li><li> Préparer le mélange réactionnel et l&#39;aliquote de 300 ul à microtubes </li><li> Réactions préincuber à 37 ° C pendant 1 minute </li><li> Démarrer réactions en ajoutant l&#39;enzyme à des intervalles spécifiques </li><li> Ajouter hydroxylamine aux mêmes intervalles pour arrêter les réactions et incuber à 60 ° C pendant 5 minutes </li><li> Ajouter le chlorure ferrique / solution d&#39;acide trichloracétique pour le développement de couleur </li><li> Centrifuger les tubes à 18.000 xg pendant 1 minute </li><li> Mesurer l&#39;absorbance à 540 nm </li><li> Déterminer la quantité de phosphate acétyl consommée par rapport à la courbe standard </li></ol>tp_upload/3474/3474fig1.jpg &quot;/&gt; Figure 1. Schéma Protocole <img alt="Figure 2" src="/files/ftp_upload/3474/3474fig2.jpg" /> Figure 2. Normes de phosphate acétyle. L&#39;augmentation des concentrations de phosphate ont été acétyl réagi avec de l&#39;hydroxylamine et la couleur a été développé comme décrit. Le contrôle manquent de phosphate acétyle est jaune vif, et les réactions contenant des quantités croissantes de phosphate acétyl sont successivement de couleur plus foncée. Ce changement de couleur est mesurée à 540 nm. <img alt="Figure 3" src="/files/ftp_upload/3474/3474fig3.jpg" /> Figure 3. Exemple de courbe de standards acétyl phosphate. Des quantités variables de phosphate ont été acétyl réagi avec la solution d&#39;hydroxylamine et développement de la couleur et l&#39;absorbance a été mesurée à 540 nm. L&#39;absorbance à 540 nm par rapport phosphate de micromoles d&#39;acétyle a été tracée et un ajustement linéaire pour les données ont été appliqués. &lt;/ P&gt; <img alt="Figure 4" src="/files/ftp_upload/3474/3474fig4.jpg" /> Figure 4. Utilisation du phosphate par l&#39;acétyl M. ATP-production acétate kinase thermophila. L&#39;activité enzymatique a été déterminée dans le sens de la formation d&#39;acétate, en présence de 5 mmol / L d&#39;ADP avec des concentrations variables de phosphate acétyle. Le montant de la consommation de phosphate acétyl a été déterminée comme la différence entre le montant de départ de phosphate acétyle dans la réaction et la quantité de phosphate acétyl restant après la réaction enzymatique. Les analyses ont été effectuées en triple avec barres d&#39;erreur s&#39;affiche. <img alt="Figure 5" src="/files/ftp_upload/3474/3474fig5.jpg" /> Figure 5. Utilisation des phosphates acétyl par le PP i-produisant E. acétate kinase histolytica. L&#39;activité enzymatique dans le sens de la formation d&#39;acétate a été déterminée en présence de 0.Phosphate de sodium 2 mol / L avec des concentrations variables de phosphate acétyle. Le montant de la consommation de phosphate acétyl a été déterminée comme la différence entre le montant de départ de phosphate acétyle dans la réaction et la quantité de phosphate acétyl restant après la réaction enzymatique. Les analyses ont été effectuées en triple avec barres d&#39;erreur s&#39;affiche.

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Pas de conflits d&#39;intérêt déclarés.

La détection de phosphate acétyle dans ce dosage est tributaire d&#39;une concentration suffisante de chlorhydrate d&#39;hydroxylamine et de la concentration et l&#39;acidité de la solution de chlorure ferrique. Altération du volume d&#39;essai, il faudra reconsidérer ces deux composantes. Les réactions enzymatiques décrites ici ont été réalisées à 37 ° C à la résiliation d&#39;hydroxylamine réalisée à 60 ° C pendant 5 minutes. Cette température élevée est essentielle pour permettre la conversion r...

<table border="1"><tr><td width="160"> Nom du réactif </td><td width="108"> Société </td><td width="134"> Numéro de catalogue </td><td width="103"> Commentaires </td></tr><tr><td> Phosphate de Acétyl </td><td> Sigma-Aldrich </td><td> 01409 </td><td> Sel de lithium (97%) </td></tr><tr><td> Phosphate monosodique (déshydratent) </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td> Phosphate de sodium dibasique (anhydre) </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td> Le chlorure de magnésium (hexahydrate) </td><td> ThermoFisher </td><td> M33 </td><td></td></tr><tr><td> Tris Base </td><td> ThermoFisher </td><td> B152 </td><td></td></tr><tr><td> Le chlorure ferrique (hexahydrate) </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td> L&#39;acide trichloracétique </td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td> Chlorhydrate d&#39;hydroxylamine </td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td> L&#39;adénosine 5&#39;-diphosphate sel de sodium </td><td> Sigma-Aldrich </td><td> A2754 </td><td></td></tr><tr><td> BIOMATE III Spectrophotomètre </td><td> ThermoFisher </td><td> 142982082 </td><td> Standard UV / VIS spectrophotomètre </td></tr></table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

Acétate kinase, un membre de l&#39;acétate et le sucre kinase-Hsp70-actine superfamille enzymatique (ASKHA) 1-5, est responsable de la phosphorylation réversible de l&#39;acétate d&#39;acétyl phosphate de l&#39;ATP en utilisant comme substrat. Acétate kinases sont omniprésents dans les bactéries, dans un genre d&#39;Archaea, et sont également présents dans les microbes de l&#39;Eukarya 6. L&#39;acétate kinase plus bien caractérisé est celui de la thermophila productrices de méthane archaeon Methanosarcina 7-14. Un acétate kinase, qui ne peut utiliser PP i, mais pas dans le sens de l&#39;ATP acétyl phosphate formant a été isolé de E. histolytica, l&#39;agent causal de la dysenterie amibienne, et a jusqu&#39;ici seulement été trouvé dans ce genre 15,16. Dans le sens de la formation de phosphate acétyle, l&#39;activité kinase de l&#39;acétate est généralement mesurée en utilisant le dosage hydroxamate, d&#39;abord décrit par Lipmann17-20, un essai de couplage dans lesquels la conversion de l&#39;ATP en ADP est couplée à l&#39;oxydation du NADH en NAD + par la pyruvate kinase enzymes et de la lactate déshydrogénase 21,22, ou un dosage mesurant la libération de phosphate inorganique après la réaction du produit de phosphate d&#39;acétyle avec de l&#39;hydroxylamine 23. Activité dans le sens opposé, de l&#39;acétate de formation direction est mesurée par le couplage formation d&#39;ATP à partir d&#39;ADP à la réduction du NADP + en NADPH par les enzymes hexokinase et glucose-6-phosphate déshydrogénase 24. Nous décrivons ici une méthode pour la détection de l&#39;activité kinase d&#39;acétate dans le sens de la formation d&#39;acétate qui ne nécessite pas d&#39;enzymes de couplage, mais est plutôt basé sur la détermination directe de la consommation de phosphate acétyle. Après la réaction enzymatique, restant de phosphate acétyle est converti en un complexe ferrique hydroxamate qui peuvent être mesurées par spectrophotométrie, comme pour le dosage hydroxamate. Ainsi, contrairement à l&#39;èreAndard test couplé à cette direction qui est tributaire de la production d&#39;ATP à partir d&#39;ADP, ce dosage direct peut être utilisé pour les kinases acétate qui produisent de l&#39;ATP ou PP i.

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Une méthode pour la détermination de l&#39;activité kinase acétate est décrite. Ce test utilise une réaction directe pour déterminer l&#39;activité enzymatique et la cinétique de l&#39;acétate kinase dans le sens de l&#39;acétate de former avec différents accepteurs phosphoryle. Par ailleurs, cette méthode peut être utilisée pour doser les autres phosphates d&#39;acétyle ou l&#39;acétyl-CoA d&#39;enzymes utilisant.

Détection directe de l&#39;activité Acétate de formation de l&#39;acétate kinase Enzyme

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

direct-detection-acetate-forming-activity-enzyme-acetate

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JoVE Journal

Biology

23.0K vues.  Clemson University. Une méthode pour la détermination de l'activité kinase acétate est décrite. Ce test utilise une réaction directe pour déterminer l'activité enzymatique et la cinétique de l'acétate kinase dans le sens de l'acétate de former avec différents accepteurs phosphoryle. Par ailleurs, cette méthode peut être utilisée pour doser les autres phosphates d'acétyle ou l'acétyl-CoA d'enzymes utilisant.

Vidéo: Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Detection of Protein Ubiquitination

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

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

La détection des protéines ubiquitination

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

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Biologie

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

L&#39;utilisation du kit de détection de protéase fluorescent pour déterminer l&#39;activité de protéase

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME (Enzyme Linked Immuno Magnetic électrochimique) Méthode de détection des mycotoxines

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

La production de transgéniques Xenopus laevis Intégration par médiation des enzymes de restriction et de la transplantation nucléaire

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Détection de l&#39;oxyde nitrique et de l&#39;anion superoxyde radical par spectroscopie de résonance paramagnétique électronique à partir de cellules en utilisant des pièges de spin

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Activités de surveillance kinase et la phosphatase dans le cycle cellulaire par Ratiométrique FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Dosage de l&#39;activité kinase de LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

GC basé sur la détection de l&#39;acétate de Aldononitrile acide dérivé de glucosamine et de muramique pour la détermination des résidus microbienne dans le sol

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Mesure de larves activité dans le<em&gt; Drosophila</em&gt; Moniteur d&#39;activité

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

Étude de l’activité des neuropeptides et d’autres régulateurs du système excréteur chez le moustique adulte

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...