Avant de commencer l&#39;essai, nous avons besoin pour s&#39;assurer que les réactifs suivants sont correctement préparées: <ol><li> Un tampon phosphate de potassium 50 mM, pH 7,5. Préparer en utilisant 11,4 mg / ml de phosphate de potassium dibasique, trihydraté dans l&#39;eau purifiée et d&#39;ajuster le pH avec HCl 1M. Cette solution est placée à 37 ° C avant utilisation. </li><li> Une solution de caséine à 0,65% en poids / volume, préparé en mélangeant 6,5 mg / ml de caséine dans le tampon phosphate de potassium 50 mM. Progressivement augmenté la température de la solution en agitant doucement à 80-85 ° C pendant environ 10 minutes jusqu&#39;à obtenir une dispersion homogène est atteint. Il est très important de ne pas faire bouillir la solution. Le pH est ensuite ajusté, si nécessaire avec NaOH et HCl. </li><li> Une solution 110 mM d&#39;acide trichloracétique, préparé en diluant un stock de 01h55 6.1N avec de l&#39;eau purifiée. L&#39;acide trichloracétique est un acide fort et doit être manipulé avec soin. </li><li> 0,5 mM Folin &amp; Ciolcaltea, ou réactif de phénol de Folin, qui est la solution qui va réagir avec la tyrosine à générer un changement de couleur mesurables qui seront directement liés à l&#39;activité des protéases. Réactif de Folin phénol est un acide et doit être manipulé avec soin. </li><li> Une solution 500 mM Sodium Carbonate, préparés en utilisant 53 mg / ml de carbonate de sodium dans l&#39;eau purifiée anyhydrous. Une solution de diluant enzyme, qui se compose de 10 mM de tampon acétate de sodium avec de l&#39;acétate de calcium 5mM, pH 7,5, à 37 ° C. Cette solution est ce que nous utilisons pour dissoudre les échantillons solides ou protéase solutions diluées enzyme. </li><li> 1,1 mM de L-tyrosine solution stock standard. Préparé en utilisant 0,2 mg / ml de L-tyrosine dans l&#39;eau purifiée et chauffée doucement jusqu&#39;à ce que la tyrosine se dissout. Comme avec la caséine, ne pas faire bouillir cette solution. Autoriser la norme de L-tyrosine refroidir à température ambiante. Cette solution sera diluée davantage pour faire de notre courbe standard. </li><li> Solution de protéase. Immédiatement avant utilisation, dissoudre dans une solution de protéase diluant d&#39;enzyme préparée à l&#39;étape 6. </li></ol> Si nécessaire, utilisez un échantillon de protéase solide de l&#39;activité prédéterminé, qui est dissous en utilisant le diluant d&#39;enzymes à 0,1-0,2 unités / ml. Cette solution sert de contrôle positif pour le test de contrôle qualité et la validation des calculs nous allons effectuer pour déterminer l&#39;activité enzymatique. Mise en place du dosage de la protéase et des courbes standard <ol><li> Pour commencer ce test, trouver des flacons adaptés qui contiendra environ 15 ml. Pour chaque enzyme qui seront testés, 4 flacons sont nécessaires. Un flacon sera utilisé comme un blanc, et trois autres seront utilisés pour l&#39;activité de dosage de trois dilutions de la protéase. Trois dilutions sont utiles lors de la vérification des calculs finale contre l&#39;autre. <ol><li> Pour chaque série de quatre flacons, ajoutez 5 ml de notre solution de caséine à 0,65%. Qu&#39;ils s&#39;équilibrer dans un bain d&#39;eau à 37 ° C pendant environ 5 minutes. </li><li> Ajouter des volumes variables de solution enzymatique qui sera testé à trois des fioles d&#39;échantillon de test, mais pas le vide. Mélanger en remuant et laisser incuber à 37 ° C pendant exactement dix minutes. L&#39;activité de la protéase et la libération de la tyrosine consécutifs pendant ce temps d&#39;incubation est de ce qui sera mesuré et comparé entre les échantillons de test. </li></ol></li><li> Après cette incubation de 10 minutes, ajouter les 5 ml du réactif TCA dans chaque tube pour arrêter la réaction. Ensuite, ajoutez un volume approprié de solution d&#39;enzyme dans chaque tube, même le blanc, de sorte que le volume final de solution d&#39;enzyme dans chaque tube est de 1 ml. Ceci est fait pour rendre compte de la valeur d&#39;absorption de l&#39;enzyme elle-même et pour s&#39;assurer que le volume final de chaque tube est égal. Incuber les solutions à 37 ° C pendant 30 minutes. Pendant cette incubation de 30 minutes, vous pouvez configurer votre tyrosine dilutions standard. Utilisez 6 flacons DRAM (DRAM flacons peuvent être remplacés par des tubes en polypropylène) qui peut facilement contenir 8 ml. Pour les six fioles, ajouter les 1,1 mM tyrosine solutions de stock standard avec les volumes suivants de la MLS: 0,05, 0,10, 0,20, 0,40, 0,50. Ne pas ajouter de toute norme de tyrosine à la vierge. Normes inférieures peuvent être nécessaires pour des échantillons de test impurs qui donneront changement de couleur peu. Une fois la solution de tyrosine standard a été ajouté, ajouter un volume approprié d&#39;eau purifiée pour chacune des normes pour porter le volume à 2 ml. </li><li> Après l&#39;incubation de 30 minutes, le filtre de chacune des solutions de test et le blanc en utilisant une seringue de 0,45 um polyéthersulfone filtre. La filtration est nécessaire pour éliminer toute insolubles à partir des échantillons. <ol><li> Ajouter la filtration 2 ml des échantillons de test et vierge filtrat à 4 flacons de DRAM qui peut contenir au moins 8 ml. Le même type de flacon dans lequel les normes ont été préparées peuvent être utilisées. </li><li> Pour tous les flacons contenant les normes et standards vierge, ajoutez 5 ml de carbonate de sodium. Pour de meilleurs résultats, ajouter 1 ml de réactif de Folin a tout de suite après. </li><li> Ajouter du carbonate de sodium à réglementer toute baisse de pH créé par l&#39;ajout du réactif de Folin l&#39;a. </li><li> Ajouter carbonate de sodium pour les échantillons à tester et TESvierge t. Ces solutions deviennent troubles après l&#39;ajout de carbonate de sodium. Ajouter le réactif de Folin l&#39;, qui va réagir principalement avec la tyrosine libre. </li><li> Mélangez les flacons dram par tourbillonnement et incuber à 37 ° C pendant 30 minutes. </li></ol> Après cette incubation, vous devriez remarquer que les normes ont une gradation de couleur en corrélation avec la quantité de tyrosine ajoutée; concentrations les plus élevées de la tyrosine sombres apparaissent. Vous pouvez également changer la couleur préavis appréciable dans nos échantillons de test. 2mls de ces solutions sont filtrées à l&#39;aide de 0,45 um polyéthersulfone filtre seringue dans des cuvettes appropriés. Maintenant que le dosage est effectué, vous pouvez procéder au spectrophotomètre pour enregistrer nos valeurs d&#39;absorbance. </li></ol> De mesurer l&#39;absorbance et de calculer l&#39;activité enzymatique <ol><li> L&#39;absorbance des échantillons est mesurée par un spectrophotomètre en utilisant une longueur d&#39;onde de 660nm. Le trajet de la lumière est réglée à 1cm. Enregistrer les valeurs d&#39;absorbance pour les normes, les standards vierge, les échantillons d&#39;essai différentes, et vierge de test. Une fois toutes les données ont été recueillies, la courbe standard peut être créé. Afin de générer la courbe, la différence d&#39;absorbance entre le blanc standard et standard doit être calculé. Il s&#39;agit de la valeur d&#39;absorbance due à la quantité de tyrosine dans les solutions standard. Après ce calcul simple, créer la courbe standard à l&#39;aide d&#39;un programme graphique pour tracer le changement d&#39;absorbance de nos normes sur l&#39;axe Y, par rapport au montant en micromoles pour chacun de nos cinq normes sur l&#39;axe X. <table style="width: 416px; height: 118px;" border="0"><tbody><tr><th style="text-align:center"> Volume de la norme Tyrosine </th><th style="text-align:center"> Tyrosine umoles </th></tr><tr><td style="text-align:center"> 0,05 </td><td style="text-align:center"> 0,055 </td></tr><tr><td style="text-align:center"> 0,10 </td><td style="text-align:center"> 0,111 </td></tr><tr><td style="text-align:center"> 0,20 </td><td style="text-align:center"> 0,221 </td></tr><tr><td style="text-align:center"> 0,40 </td><td style="text-align:center"> 0,442 </td></tr><tr><td style="text-align:center"> 0,50 </td><td style="text-align:center"> 0,553 </td></tr></tbody></table></li><li> Après les points de données ont été entrées, générer une ligne de meilleur ajustement et l&#39;équation de pente correspondant. Trouver le changement d&#39;absorbance dans les échantillons de test en calculant la différence entre l&#39;absorbance de l&#39;échantillon de test et l&#39;absorbance du blanc d&#39;essai. Insertion de la valeur d&#39;absorbance de l&#39;un des échantillons d&#39;essai dans l&#39;équation de pente et la résolution de se traduira par la micromoles de tyrosine libérée au cours de cette réaction protéolytique particulière. Pour obtenir de l&#39;activité de l&#39;enzyme en unités par / ml, effectuer le calcul suivant: (Umole équivalents tyrosine libérée) x (11) Unités / ml = Enzyme __________________________________________ (1) x (10) x (2) 11 = volume total (en millilitres) de dosage 10 = Temps d&#39;essai (en minutes) selon la définition Unité 1 = Volume d&#39;enzyme (en millilitres) d&#39;enzyme utilisée 2 = Volume (en millilitres) utilisées dans la détermination colorimétrique Prenez le nombre d&#39;équivalents micromoles tyrosine libérée obtenus à partir de l&#39;équation de pente et le multiplier par le volume total de l&#39;essai dans la MLS, qui dans notre cas est 11mls. Diviser cette valeur par trois autres quantités: le temps de l&#39;essai, ce qui nous avons couru pendant 10 minutes, le volume d&#39;enzyme utilisé dans le dosage, qui était variée (nous allons utiliser 1ml), le volume de millilitres utilisés dans la détection colorimétrique, ce qui peut diffèrent en fonction de votre cuvette. Nous avons utilisé 2 ml. </li><li> Micromoles de la tyrosine, divisé par le temps en minutes rendements mesure de l&#39;activité de la protéase appelée «unités». Nous pouvons annuler les unités de mesure de volume dans le numérateur et le dénominateur, laissant un measurment de l&#39;activité enzymatique en termes d&#39;unités / ml. Afin de déterminer l&#39;activité dans un échantillon de protéase solides diluées dans du diluant d&#39;enzymes, nous divisons notre activité en unités / ml par la concentration de solides utilisés dans cet essai à l&#39;origine en mg / ml., Nous laissant avec l&#39;activité en termes d&#39;unités / mg . Unités / ml enzyme Unités / mg de solide = _____________________ mg de solide / enzyme ml </li></ol>

<ol>
	<li>Anson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Gen. Physiol. 22, 79-89 (1938).</li><li>Folin, O., Ciocalteau, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Biol. Chem. 73, 627 (1929).</li></ol>

Nous venons de vous montrer comment analyser l&#39;activité de la protéase à l&#39;aide de Sigma test universelle activité de la protéase. En outre, ce test est utile pour s&#39;assurer que nos protéases ont déterminé avec précision l&#39;activité avant de les recevoir pour vos expériences. Comme vous l&#39;avez vu, lors de cette procédure, il est d&#39;une importance primordiale à retenir la chaleur à la fois de la caséine et des solutions de tyrosine lentement et de ne pas les faire bouillir. Aussi, il est essentiel de préparer les blancs différents pour les deux normes de votre et pour chaque échantillon d&#39;essai que vous avez.

<table>
	<tbody>
		<tr>
			<td>Protease</td>
			<td>Sigma-Aldrich</td>
			<td>P4630</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Dibasic, Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>P5504</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>T0699</td>
			<td></td>
		</tr>
		<tr>
			<td>Folin &#38; Ciocalteu&#39;s Phenol Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>F9252</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate, Anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate, Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S8625</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>C1000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine, Free Base</td>
			<td>Sigma-Aldrich</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Profiler Kit</td>
			<td>Sigma-Aldrich</td>
			<td>PP0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Fluorescent Detection Kit</td>
			<td>Sigma-Aldrich</td>
			<td>PF0100</td>
			<td></td>
		</tr>
	</tbody>
</table>

sigma's non-specific protease activity assay - casein as a substrate

Les protéases casser les liaisons peptidiques. Dans le laboratoire, il est souvent nécessaire de mesurer et / ou comparer l&#39;activité des protéases. Sigma non spécifiques dosage de l&#39;activité de la protéase peut être utilisé comme une procédure normalisée pour déterminer l&#39;activité des protéases, qui est ce que nous faisons lors de nos procédures de contrôle qualité. Dans cet essai, la caséine agit comme un substrat. Lorsque la protéase nous testons digère la caséine, l&#39;acide aminé tyrosine est libéré avec d&#39;autres acides aminés et fragments peptidiques. Réactif de Folin et Ciocalteus phénol, ou Folin réagit principalement avec la tyrosine libre de produire un chromophore de couleur bleue, qui est quantifiable et mesurée comme une valeur d&#39;absorbance sur le spectrophotomètre. Le plus tyrosine qui est libérée à partir de caséine, plus les chromophores sont générés et le plus fort de l&#39;activité de la protéase. Les valeurs d&#39;absorbance généré par l&#39;activité de la protéase sont comparées à une courbe standard, qui est généré par la réaction des quantités connues de la tyrosine avec le réactif CF de corréler les changements d&#39;absorbance avec le montant de la tyrosine en micromoles. De la courbe standard de l&#39;activité de la protéase échantillons peut être déterminé en termes de parts, qui est le montant en micromoles d&#39;équivalents tyrosine libéré de la caséine par minute. Pour voir cet article en chinois, <a href="http://jove.com/index/details.stp?ID=910">cliquez ici</a>

Watch this Scientific Journal Video about Sigma's Non-specific Protease Activity Assay - Casein as a Substrate at JoVE.com

Sigma&#039;s Non-specific Protease Activity Assay - Casein as a Substrate

Les protéases casser les liaisons peptidiques. Dans le laboratoire, il est souvent nécessaire de mesurer et / ou comparer l&#39;activité des protéases. Sigma non spécifiques dosage de l&#39;activité de la protéase peut être utilisé comme une procédure normalisée pour déterminer l&#39;activité des protéases.

Sigma non spécifiques Assay activité de la protéase - caséine comme substrat

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.  Sigma's non-specific protease ...

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Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

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Vidéo: Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Génotypage rapide des tissus de souris en utilisant Sigma Extract-N-Amp PCR Kit Tissu

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

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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 ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Un test pour mesurer l&#39;activité des Escherichia coli Inductible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Un test basé sur Matrigel Tube Formation d&#39;évaluer l&#39;activité Vasculogenic de cellules tumorales

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

Flux de travail d&#39;étiquetage protéase et catalysée par un acide Embauche<sup&gt; 18</sup&gt; O enrichi en eau

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Souris îlot de Langerhans isolement à l&#39;aide d&#39;une combinaison de la collagénase purifiée et protéase neutre

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

Détermination Membrane Protein topologie utilisant la fluorescence protection protéase (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Comet Assay comme une mesure indirecte de stress oxydatif systémique

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identification de paires Kinase-substrat en utilisant criblage à haut débit


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.