Instrucciones de preparación El tampón de incubación, tampón de análisis y solución de 0,6 N TCA son siempre tan dispuestos a utilizar las soluciones. El sustrato de FITC-caseína - Se recomienda a la alícuota del sustrato FITC-caseína en volúmenes más pequeños al llegar a evitar una repetición de los ciclos de congelación y descongelación. Si el sustrato FITC-caseína es sometido a repetidos ciclos de congelación y descongelación, un ligero aumento en el fondo va a ocurrir, por lo tanto, la reducción de la sensibilidad. Las alícuotas deben ser almacenadas a -20 ° C y protegido de la luz. Cada muestra, la reacción en blanco, o el control requiere de 20 l de sustrato de la FITC-caseína. Congelación / descongelación, así como una mezcla vigorosa o agitación puede hacer que el FITC se separe de la caseína que resulta en un fondo más alto de lectura y causa menos sustrato para estar disponible para la escisión de la proteasa. El isotiocianato de fluoresceína (FITC) de solución de control: el control FITC puede ser reconstituida en el tampón de ensayo a la concentración adecuada. Esta solución debe ser fresco y protegido de la luz. Final de la tripsina solución de control - añadir 100 ml de HCl 1 mM en el vial de la tripsina, la proteasa de control (código de producto T 6567). Mezclar brevemente para asegurar la tripsina se disuelve. Añadir 900 l de la incubación de amortiguación y mezclar bien. Por otra parte, los tampones se pueden utilizar otros si así lo desea para el ensayo. La concentración final de trabajo de la tripsina es de 20 mg / l. Cuando esté listo para preparar la solución de control de la tripsina, combine una parte alícuota de la solución de tripsina ácida con la cantidad correcta del tampón de incubación (1 tripsina acidificada parte a 9 partes de búfer). Almacenamiento de la tripsina en condiciones ácidas aumenta la estabilidad de la tripsina. La solución de control de la tripsina es sensible a la temperatura y no es estable a bajas concentraciones. Por favor revise el boletín técnico del kit para el almacenamiento y la estabilidad de las soluciones preparadas. Procedimiento <ol><li> Es más fácil preparar los controles, los espacios en blanco, y todas las muestras a la vez. Para cada muestra, se añaden 20 l de tampón de incubación, 20 l de FITC-caseína sustrato, y 10 l de la muestra a un tubo de microcentrífuga. Para muestras con actividad de proteasa elevada, dilución de la muestra puede ser necesaria. </li><li> Preparación de muestras de control adecuadas (ver muestras de control) mediante la adición de 20 l de tampón de incubación, 20 l de FITC-caseína sustrato, y 10 l de la muestra de control a un tubo de microcentrífuga. </li><li> Preparar una muestra en blanco por la adición de 20 l de tampón de incubación, 20 l de FITC-caseína sustrato, y 10 l de agua ultrapura para un tubo de microcentrífuga. </li><li> Mezclar suavemente cada tubo y se incuba a 37 ° C en la oscuridad durante 60 minutos. Tenga cuidado de no mezclar con demasiada fuerza, como la turbulencia excesiva puede provocar la fluorescencia de fondo de alta y reducir la sensibilidad de la prueba. Nota: El tiempo de incubación puede extenderse hasta 24 horas para aumentar la sensibilidad. Tenga cuidado de no exceder de 24 horas como el FITC-caseína puede empezar a degradarse, lo que la fluorescencia de fondo de alta. </li><li> Después de la incubación añadir 150 ml de la solución 0,6 N de ácido tricloroacético a cada tubo de microcentrífuga. TCA es muy corrosivo y debe ser manejado mientras esté usando el equipo de protección adecuado. </li><li> Mezclar suavemente e incubar a 37 ° C en la oscuridad durante 30 minutos. </li><li> Centrifugar los tubos durante 10 minutos a 10.000 x g. El sobrenadante contiene el ácido soluble en fragmentos, marcado con FITC y se utiliza para la medición de fluorescencia. </li></ol> Las mediciones de fluorescencia Estos métodos se pueden escalar hacia arriba o abajo de acuerdo a los requisitos de la instrumentación disponible. Por comparación con una curva estándar preparada con las muestras de control adecuadas, restar la lectura de fluorescencia de la muestra en blanco (FLUblank) del valor de cada muestra (FLUtest). Cubetas <ol><li> Pipeta de 10 l de sobrenadante (paso 7) y 1 ml de tampón de ensayo en una cubeta adecuada y mezclar suavemente. Nota: La solución del tampón de ensayo sobrenadante y se pueden almacenar en la oscuridad a 2-8 ° C hasta 24 horas antes de la medición de la fluorescencia. </li><li> Registro de la intensidad de fluorescencia con excitación a 485 nm y el control de la longitud de onda de emisión de 535 nm. </li></ol> Placas de pocillos múltiples <ol><li> Pipeta de 10 l de la Nota sobrenadante (paso 7) y 1 ml de tampón de ensayo en un tubo adecuado o vial y mezclar suavemente: La solución del tampón de ensayo sobrenadante y se pueden almacenar en la oscuridad a 2-8 ° C hasta 24 horas antes de la medición de la fluorescencia. </li><li> Transferencia de 200 l en un pozo negro de una placa de 96 pocillos. Registro de la intensidad de fluorescencia con excitación a 485 nm y el control de la longitud de onda de emisión de 535 nm. O </li></ol><ol><li> Pipetear 2 l de sobrenadante (paso 7) y 200 l de la B Ensayouffer en un pozo negro de una placa de 96 pocillos. Nota: La solución del tampón de ensayo sobrenadante y se pueden almacenar en la oscuridad a 2-8 ° C hasta 24 horas antes de la medición de la fluorescencia. </li><li> Registro de la intensidad de fluorescencia con excitación a 485 nm y el control de la longitud de onda de emisión de 535 nm. </li></ol> Las muestras de control La solución de control de tripsina se puede utilizar para confirmar la prueba está funcionando correctamente, para determinar el límite de detección, o crear una curva estándar general. Para el ensayo de una proteasa diferentes y específicos, se recomienda preparar una solución de control que contiene la proteasa específica en el tampón de incubación apropiado. El límite de detección del ensayo es la cantidad de la proteasa, que produce una lectura significativa de fluorescencia por encima del valor obtenido con la muestra en blanco. El límite de detección pueden variar dependiendo de la sensibilidad de la instrumentación. Diluciones seriadas de la solución de control de tripsina puede ser utilizado para generar las soluciones de control. A igualdad de la lectura al 120% del valor obtenido con la muestra en blanco se considera significativo. Habitualmente, un límite de detección de 5 ng de tripsina se obtuvo con este procedimiento. Se recomienda que al menos un 5 ng de control de la tripsina se ejecute con cada ensayo. Una solución de control de tripsina con una concentración de 0,5 mg / ml daría lugar a la deseada 5 ng de tripsina en el ensayo. Una dilución de 40 veces de la solución de control de la tripsina (20 ug / ml) resulta en una solución de control de 0,5 mg / ml, es decir, una parte de la solución de control de tripsina a 39 partes de tampón de incubación. <img src="/files/ftp_upload/1514/figure1.jpg" alt="Figura 1" /> Figura 1 (curva estándar de actividad de la tripsina): La solución de control de tripsina (20 ug / ml) también se puede utilizar para generar una curva estándar al hacer diluciones en serie (ver Figura 1). La lectura de la fluorescencia de cada muestra de control se corrigió restando la lectura de la fluorescencia de la muestra en blanco (FLUblank) del valor de cada muestra de control (FLUcontrol). Típica curva estándar de tripsina (Código de producto T 6567) con el kit y las muestras de control que van desde 0,15 mg / ml (1,5 ng) a 2,5 mg / ml (25 ng). Esta curva se generó mediante el procedimiento descrito, con un tiempo de incubación de 30 minutos para el paso 4. Medidas de fluorescencia se realizaron en una placa de pocillos múltiples. Isotiocianato de fluoresceína se presenta como un control para la calibración de los instrumentos posibles (vea las instrucciones del proveedor) o la determinación del rango de linealidad de la señal de FITC.

<ol>
	<li>Twining, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+Isothiocyanate-Labeled+Casein+Assay+for+Proteolytic+Enzymes.">Fluorescein Isothiocyanate-Labeled Casein Assay for Proteolytic Enzymes.</a> Anal. Biochem. 143, 30-34 (1984).</li></ol>

El autor es un empleado de Sigma Aldrich, que produce los reactivos y las herramientas utilizadas en este artículo.

Acabamos de mostrar un ensayo ejemplo usando el kit de la proteasa detección fluorescente para detectar la actividad de la proteasa en una muestra biológica. Este kit ha sido optimizado para detectar una amplia variedad de proteasas que se encuentran en las aplicaciones fisiológicas. Es adecuado para la detección de la serina, la cisteína, Metallo, y proteasas aspártico, sin embargo, las modificaciones pueden ser necesarias para detectar algunas proteasas específicas.

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments (optional)</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=PF0100|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12346">Protease Fluorescent Detection Kit</a></td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>PF0100</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=H1758|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12347">Hydrochloric Acid</a></td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>H1758</td>
<td>&nbsp;</td>
</table>

use of the protease fluorescent detection kit to determine protease activity

El kit de la proteasa detección fluorescente proporciona listas para el uso de reactivos para detectar la presencia de actividad de la proteasa. Este ensayo simple para detectar la actividad de la proteasa utiliza caseína marcada con isotiocianato de fluoresceína (FITC) como sustrato. Resultados de la proteasa actividad en la escisión del sustrato de caseína marcada con FITC en fragmentos más pequeños, que no precipitan en condiciones ácidas. Después de la incubación de la muestra de la proteasa y el sustrato, la reacción se acidifica con la adición de ácido tricloroacético (TCA). La mezcla se centrifuga con el sustrato sin digerir formar una bolita y los más pequeños, fragmentos de ácido soluble que permanece en solución. El sobrenadante se neutraliza y la fluorescencia de la FITC fragmentos se mide. El procedimiento descrito kit detecta el control de la proteasa tripsina a una concentración de aproximadamente 0,5 mg / ml (5 ng de tripsina añadido a la prueba). Esta sensibilidad se puede aumentar con un tiempo de incubación más largo, hasta 24 horas. El ensayo se realiza en tubos de microcentrífuga y los procedimientos se proporcionan para la detección de fluorescencia utilizando cubetas o placas multipocillo.

Watch this Scientific Journal Video about Use of the Protease Fluorescent Detection Kit to Determine Protease Activity at JoVE.com

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

El kit de detección fluorescente de la proteasa se ha diseñado para la medición de la actividad de la proteasa con fluorometría. También es adecuado para la detección de trazas de contaminación de la proteasa. El método se basa en la hydroysis proteolítica de una fórmula propia de un sustrato marcado con FITC caseína.

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 ...

use-protease-fluorescent-detection-kit-to-determine-protease

Nanyang Technological University

Research

JoVE Journal

Biology

28.1K vistas.  Sigma Aldrich. El kit de detección fluorescente de la proteasa se ha diseñado para la medición de la actividad de la proteasa con fluorometría. También es adecuado para la detección de trazas de contaminación de la proteasa. El método se basa en la hydroysis proteolítica de una fórmula propia de un sustrato marcado con FITC caseína.

Video: Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Purificación del ADN plásmido de las colonias de bacterias Utilizando el kit de Qiagen Miniprep

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Investigación

Biología

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Inmunohistoquímica: las secciones de parafina utilizando la Vectastain ABC Kit de Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etiquetado de las células madre con tintes fluorescentes para la detección no invasiva con imágenes ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

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

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 activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.
To view this article in Chinese, <a href="https://jove-com.remotexs.ntu.edu.sg/index/details.stp?ID=910">click here</a>

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 activity assay may be used as a standardized procedure to determine the activity of proteases.

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 ...

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 detección de ubiquitinación de proteínas

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

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

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

La detección directa de la actividad de acetato de formación de la enzima cinasa de acetato

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

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Criopreservación de espermatozoides de ratón y de recuperación con el I · Kit de Cryo

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

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 ...