Antes de comenzar el ensayo, hay que asegurarse de que los siguientes reactivos se preparan correctamente: <ol><li> Una de potasio 50 mM tampón fosfato, pH 7,5. Prepare con 11,4 mg / ml de fosfato de potasio dibásico, trihidrato en agua purificada y ajustar el pH con HCl 1M. Esta solución se coloca a 37 ° C antes de su uso. </li><li> Un 0,65% peso / volumen caseína solución preparada mezclando 6,5 mg / ml de caseína en el tampón fosfato 50 mM de potasio. Poco a poco aumentó la temperatura de la solución con agitación suave a 80-85 º C durante unos 10 minutos hasta una dispersión homogénea se logra. Es muy importante que no hierva la solución. El pH se ajusta si es necesario con NaOH y HCl. </li><li> A 110 mM solución de ácido tricloroacético, preparado por dilución de una 1:55 acciones 6.1N con agua purificada. El ácido tricloroacético es un ácido fuerte y debe ser manejado con cuidado. </li><li> 0,5 mM de Folin y Ciolcaltea, o reactivo Folin Fenol, que es la solución que reacciona con la tirosina para generar un cambio de color mensurables que se relaciona directamente con la actividad de las proteasas. Reactivo Folin fenol es un ácido y debe manejarse con cuidado. </li><li> A 500 mM de carbonato de sodio solución preparada con 53 mg / ml de carbonato de sodio anyhydrous en agua purificada. Una solución diluyente enzima, que consta de 10 mM de tampón acetato de sodio con acetato de calcio 5 mM, pH 7,5, a 37 ° C. Esta solución es lo que usamos para disolver las muestras sólidas de la proteasa o soluciones diluidas de la enzima. </li><li> 1,1 mM de L-tirosina Solución madre del estándar. Elaborado a partir de 0,2 mg / ml de L-tirosina en agua purificada y se calienta suavemente hasta que la tirosina se disuelve. Al igual que con la caseína, no hervir esta solución. Que la norma L-tirosina para enfriar a temperatura ambiente. Esta solución se diluye aún más para hacer que nuestra curva estándar. </li><li> Proteasa solución. Inmediatamente antes de su uso, se disuelven en la solución diluyente de la proteasa enzimas preparada en el paso 6. </li></ol> Si es necesario, utilice una muestra sólida de la proteasa actividad predeterminada, que se disuelve con un diluyente de la enzima a 0,1-0,2 unidades / ml. Esta solución sirve como un control positivo para el ensayo de control de calidad y la validación de los cálculos que se realizan para determinar la actividad enzimática. Configuración del ensayo de la proteasa y las curvas de calibración <ol><li> Para comenzar este ensayo, encontramos viales adecuados que contendrá alrededor de 15 ml. Para cada enzima que se pondrá a prueba, 4 viales son necesarios. Un vial se utilizará como un espacio en blanco, y otros tres se utilizará para ensayar la actividad de tres diluciones de la proteasa. Tres diluciones son útiles en la comprobación final de los cálculos el uno contra el otro. <ol><li> Para cada conjunto de cuatro viales, añadir 5ml de nuestra solución de caseína al 0,65%. Dejar que se equilibre en un baño de agua a 37 ° C durante unos 5 minutos. </li><li> Añadir la variación de volúmenes de solución de enzima que se pondrá a prueba a tres de los viales de prueba de muestra, pero no el blanco. Mezclar por agitación e incubar a 37 ° C durante diez minutos exáctamente. La actividad de la proteasa y la liberación consiguiente de la tirosina durante este tiempo de incubación es de lo que va a ser medido y comparado entre las muestras de prueba. </li></ol></li><li> Después de esta incubación de 10 minutos, agregue el 5 ml del reactivo de TCA a cada tubo para detener la reacción. A continuación, agregue un volumen adecuado de solución de enzima a cada tubo, hasta el blanco, por lo que el volumen final de solución de enzima en cada tubo es de 1 ml. Esto se hace para tener en cuenta el valor de absorción de la enzima en sí y para asegurarse de que el volumen final en cada tubo es igual. Se incuban las soluciones a 37 ° C durante 30 minutos. Durante esta incubación de 30 minutos, es posible que desee configurar su diluciones estándar de tirosina. Use 6 viales DRAM (DRAM viales pueden ser sustituidos por tubos de polipropileno) que puede contener 8 ml. A los seis viales, añadir los 1,1 mM soluciones de tirosina de patrón con los siguientes volúmenes de la MLS: 0,05, 0,10, 0,20, 0,40, 0,50. No agregue ningún estándar de tirosina en el espacio en blanco. Niveles más bajos puede ser necesaria para las muestras de prueba impuros que los cambios sean poco de color. Una vez que la solución estándar de tirosina se ha añadido, añadir una cantidad adecuada de agua purificada a cada una de las normas para obtener un volumen de 2 ml. </li><li> Después de la incubación de 30 minutos, filtrar cada una de las soluciones de prueba y el espacio en blanco con un 0,45 m de polietersulfona filtro de jeringa. La filtración es necesaria para eliminar cualquier insolubles de las muestras. <ol><li> Añadir 2 ml de la filtración de las muestras de ensayo y en el filtrado de 4 viales de DRAM que puede contener un mínimo de 8 ml. El mismo tipo de vial en el que las normas fueron preparadas se pueden utilizar. </li><li> A todos los viales que contienen las normas y estándares en blanco, agregar 5ml de carbonato de sodio. Para obtener mejores resultados, añadir 1 ml de reactivo Folin inmediatamente después. </li><li> Agregar carbonato de sodio para regular cualquier disminución del pH creado por la adición de reactivo Folin de la. </li><li> Agregar carbonato de sodio a las muestras de prueba y test en blanco. Estas soluciones se nublan después de la adición de carbonato de sodio. Añadir el reactivo Folin es el que va a reaccionar principalmente con tirosina libre. </li><li> Mezclar los viales dram por agitación e incubar a 37 ° C durante 30 minutos. </li></ol> Después de esta incubación, se debe notar que las normas tienen una gradación de color en correlación con la cantidad de tirosina añadido, las mayores concentraciones de tirosina que aparecen más oscuras. También puedes notar el cambio de color apreciable en las muestras de nuestra prueba. 2mls de estas soluciones se filtran utilizando una jeringa de 0,45 m de polietersulfona filtro en las cubetas adecuadas. Ahora que se realiza el ensayo, se puede proceder a la espectrofotómetro para grabar nuestros valores de absorbancia. </li></ol> Medir la absorbancia y cálculo de la actividad enzimática <ol><li> La absorbancia de las muestras se mide con un espectrofotómetro con una longitud de onda de 660 nm. La trayectoria de la luz se encuentra a 1 cm. Registre los valores de absorbencia de los estándares, en blanco estándar, las muestras de prueba diferentes, y en blanco de ensayo. Una vez que todos los datos han sido recogidos, la curva estándar se pueden crear. Con el fin de generar la curva, la diferencia en la absorbancia entre el blanco estándar y debe ser calculado. Este es el valor de absorbancia atribuible a la cantidad de tirosina en las soluciones estándar. Después de este cálculo simple, crear la curva estándar utilizando un programa de graficación para trazar el cambio de absorbancia de los estándares en el eje Y, frente a la cantidad de micromoles por cada uno de los cinco niveles en el eje X. <table style="width: 416px; height: 118px;" border="0"><tbody><tr><th style="text-align:center"> Volumen de estándar tirosina </th><th style="text-align:center"> uMoles tirosina </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> Después de los puntos de datos han sido ingresados, generará una línea de mejor ajuste y la ecuación de la pendiente correspondiente. Buscar el cambio de absorbancia en las muestras de ensayo mediante el cálculo de la diferencia entre la absorbancia de la muestra de ensayo y la absorbancia del blanco de prueba. Insertar el valor de absorción de una de las muestras de prueba en la ecuación de la pendiente y la resolución dará lugar a la micromoles de tirosina liberada en esta reacción proteolítica particular. Para obtener la actividad de la enzima en unidades por / ml, realice el siguiente cálculo: (Umole equivalentes de tirosina liberada) x (11) Unidades / ml Enzima = __________________________________________ (1) x (10) x (2) 11 = Total de volumen (en mililitros) de ensayo 10 = Tiempo de ensayo (en minutos) de acuerdo con la definición de unidad 1 = Volumen de la enzima (en mililitros) de la enzima que se utiliza 2 = volumen (en mililitros) utilizados en la determinación colorimétrica Tome el número de equivalentes de tirosina liberada micromoles obtenidos a partir de la ecuación de la pendiente y se multiplica por el volumen total de la prueba en la MLS, que en nuestro caso es 11mls. Dividir este valor por tres otras cantidades: el tiempo del ensayo, que duró 10 minutos, el volumen de enzima utilizada en el ensayo, que fue variado (vamos a usar 1 ml), el volumen de mililitros utilizados en la detección colorimétrica, que puede variar en función de su cubeta. Se utilizó 2 ml. </li><li> Micromoles de tirosina dividido por el tiempo en minutos de medición de los rendimientos de la actividad de la proteasa llamado &quot;unidades&quot;. Podemos cancelar las unidades de medida de volumen en el numerador y el denominador, dejando una de Medidas de la actividad enzimática en términos de unidades / ml. Con el fin de determinar la actividad en una muestra de la proteasa sólido diluido en un diluyente de la enzima, dividimos nuestra actividad en las unidades / ml de la concentración de sólidos utilizados en este ensayo originalmente en mg / ml., Dejándonos con la actividad en términos de unidades / mg . Unidades / ml de enzima Unidades / mg sólido = _____________________ mg sólidos / ml enzima </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>

Acabamos mostrado cómo analizar la actividad de la proteasa utilizando universales de Sigma ensayo de actividad de la proteasa. Además, este ensayo es útil para asegurar que nuestros proteasas se han determinado con precisión la actividad antes de que usted los reciba para sus experimentos. Como se ha visto, cuando se hace este procedimiento, es de suma importancia recordar que el calor tanto de la caseína y las soluciones de tirosina lentamente y no a hervir. Además, es fundamental para preparar los espacios en blanco diferente para ambos sus estándares y para cada muestra de prueba que usted tiene.

<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

Las proteasas rompen los enlaces peptídicos. En el laboratorio, a menudo es necesario medir y / o comparar la actividad de las proteasas. No específico de Sigma ensayo de actividad de la proteasa puede ser utilizado como un procedimiento normalizado para determinar la actividad de las proteasas, que es lo que hacemos en nuestros procedimientos de control de calidad. En este ensayo, la caseína actúa como sustrato. Cuando la proteasa que estamos probando digiere la caseína, el aminoácido tirosina es liberado junto con otros aminoácidos y fragmentos peptídicos. Reactivo de Folin y Ciocalteus fenol, o Folin es principalmente reacciona con la tirosina libre para producir un cromóforo de color azul, que es cuantificable y se mide como un valor de absorbancia en el espectrofotómetro. La tirosina más que se libera de la caseína, más los cromóforos que se generan y más fuerte es el de la actividad de la proteasa. Los valores de absorción que genera la actividad de la proteasa se compara con una curva patrón, que se genera mediante la reacción de cantidades conocidas de la tirosina con el reactivo FC correlacionar los cambios en la absorbancia con la cantidad de tirosina en micromoles. De la curva estándar de la actividad de las muestras de la proteasa se puede determinar en términos de unidades, que es la cantidad de micromoles de equivalentes de tirosina liberada de la caseína por minuto. Para ver este artículo en chino, <a href="http://jove.com/index/details.stp?ID=910">haga clic aquí</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

Las proteasas rompen los enlaces peptídicos. En el laboratorio, a menudo es necesario medir y / o comparar la actividad de las proteasas. No específico de Sigma ensayo de actividad de la proteasa puede ser utilizado como un procedimiento normalizado para determinar la actividad de las proteasas.

No específico de Sigma Ensayo de actividad de la proteasa - La caseína como sustrato

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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Video: 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.

Genotipificación rápida de tejido de ratón utilizando extracto-N-Amp tejido Sigma 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 ...

Investigación

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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.

El uso del kit de la proteasa detección fluorescente para determinar la actividad de la proteasa

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 ensayo para medir la actividad de Escherichia coli Inducible lisina 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.

A Matrigel basado en tubo de ensayo de formación para evaluar la actividad de las células tumorales vasculogénica

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.

Proteasa y el ácido catalizado-Flujos de trabajo de rotulación Emplear<sup&gt; 18</sup&gt; O-agua enriquecida

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.

Aislamiento de ratón Islet Langerhans utilizando una combinación de colagenasa purificada y proteasa neutra

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.

La determinación de la proteína de membrana de topología usando fluorescencia de Protección de la proteasa (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.

Ensayo Cometa como una medida indirecta de estrés oxidativo sistémico

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.

Identificación de los pares quinasa-sustrato Usando selección de alto rendimiento


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.