Instruções de preparação O tampão de incubação, Tampão de Ensaio, e 0,6 N Solução TCA são fornecidos como ready-to-use soluções. O substrato FITC-caseína - Recomenda-se a alíquota do substrato FITC-caseína em volumes menores no momento da chegada para evitar repetidos ciclos de congelamento e descongelamento. Se o substrato FITC-caseína é submetido a repetidos ciclos de congelamento e descongelamento, um ligeiro aumento no fundo irá ocorrer, assim, diminuindo a sensibilidade. As alíquotas devem ser armazenadas a -20 ° C e protegida da luz. Cada amostra, a reação em branco, ou controle necessita de 20 mL do substrato FITC-caseína. Congelamento / descongelamento, bem como vigorosa mistura ou agitação podem fazer com que o FITC a se separar da caseína, resultando em uma maior fundo de leitura e causando menos substrato para estar disponível para a clivagem da protease. O isotiocianato de fluoresceína (FITC) Controle Solution-FITC O controle pode ser reconstituída em Tampão de Ensaio para a concentração adequada. Esta solução deve ser feita fresco e protegido da luz. Final da Solução de Controle Tripsina - Adicione 100 ml de HCl 1 mM para o frasco de tripsina, protease Control (Código do Produto T 6567). Misture rapidamente para garantir a tripsina é dissolvido. Adicionar 900 l do tampão de incubação e misture bem. Alternativamente, buffers podem ser utilizados outros se desejado para o ensaio. A concentração final de trabalho da tripsina é de 20 mg / mL. Quando estiver pronto para preparar a Solução de Controle Tripsina, combine uma alíquota da solução de tripsina ácidos com a quantidade correta do tampão de incubação (1 tripsina acidificados parte para nove partes de buffer). Armazenar a tripsina em meio ácido aumenta a estabilidade da tripsina. A solução de controle de tripsina é sensível à temperatura e não é estável em baixas concentrações. Reveja boletim técnico do kit para armazenamento e estabilidade destas soluções preparadas. Procedimento <ol><li> É mais fácil preparar controles, blanks, e amostras de uma só vez. Para cada amostra, adicionar 20 mL de tampão de incubação, 20 l de FITC-caseína de substrato, e 10 ml da amostra para um tubo de microcentrífuga. Para amostras com atividade de protease alto, diluição da amostra pode ser necessária. </li><li> Prepare amostras de controlo adequado (ver amostras de controle), adicionando 20 l de tampão de incubação, 20 l de FITC-caseína de substrato, e 10 ml da amostra de controlo a um tubo de microcentrífuga. </li><li> Prepare uma amostra em branco, adicionando 20 l de tampão de incubação, 20 l de FITC-caseína de substrato, e 10 ml de água ultrapura para um tubo de microcentrífuga. </li><li> Homogeneizar cada tubo e incubar a 37 ° C no escuro por 60 minutos. Tenha cuidado para não misturar muito vigorosamente, como turbulência excessiva pode causar fundo de fluorescência de alta e reduzir a sensibilidade do ensaio. Nota: O tempo de incubação pode ser prolongado até 24 horas para aumentar a sensibilidade. Tenha cuidado para não exceder as 24 horas como o FITC-caseína podem começar a se degradar, levando ao fundo de fluorescência de alta. </li><li> Após a incubação adicionar 150 mL da solução de ácido 0,6 N tricloroacético a cada tubo de microcentrífuga. TCA é muito corrosivo e deve ser tratado enquanto usava o equipamento de protecção adequado. </li><li> Homogeneizar e incubar a 37 ° C no escuro por 30 minutos. </li><li> Centrifugue os tubos durante 10 minutos a 10.000 x g. O sobrenadante contém o ácido solúvel, FITC fragmentos rotulados e é utilizado para a medição de fluorescência. </li></ol> Medidas de fluorescência Estes métodos podem ser ampliados ou reduzidos de acordo com os requisitos da instrumentação disponível. Para comparação com uma curva padrão preparada com as amostras de controlo adequadas, subtrair a leitura de fluorescência da amostra em branco (FLUblank) do valor de cada amostra (FLUtest). Cubetas <ol><li> Pipetar 10 mL do sobrenadante (passo 7) e 1 ml de Tampão de Ensaio numa cuvete adequado e misture delicadamente. Nota: A solução de Tampão de Ensaio sobrenadante e podem ser armazenadas no escuro a 2-8 ° C por até 24 horas antes de medir a fluorescência. </li><li> Registrar a intensidade de fluorescência com excitação em 485 nm e monitoramento do comprimento de onda de emissão de 535 nm. </li></ol> Placas com vários poços <ol><li> Pipetar 10 mL do sobrenadante Nota (passo 7) e 1 ml de Tampão de Ensaio em um tubo ou frasco adequado e misture delicadamente: A solução de Tampão de Ensaio sobrenadante e podem ser armazenadas no escuro a 2-8 ° C por até a 24 horas antes de medir a fluorescência. </li><li> Transferência de 200 mL para um poço de uma placa de 96 preto também. Registrar a intensidade de fluorescência com excitação em 485 nm e monitoramento do comprimento de onda de emissão de 535 nm. Ou </li></ol><ol><li> Pipetar 2 mL do sobrenadante (etapa 7) e 200 mL do B Assayuffer em um poço de uma placa de 96 preto também. Nota: A solução de Tampão de Ensaio sobrenadante e podem ser armazenadas no escuro a 2-8 ° C por até 24 horas antes de medir a fluorescência. </li><li> Registrar a intensidade de fluorescência com excitação em 485 nm e monitoramento do comprimento de onda de emissão de 535 nm. </li></ol> As amostras de controle A Solução de Controle de tripsina pode ser usado para confirmar o teste está funcionando adequadamente, para determinar o limite de detecção, ou criar uma curva de norma geral. Para o ensaio de uma protease, diferentes específico, recomenda-se para preparar uma solução de controle que contém a protease específica no buffer de incubação apropriado. O limite de detecção do ensaio é a quantidade de protease, que produz uma leitura significativa de fluorescência acima do valor obtido com a amostra em branco. O limite de detecção irá variar dependendo da sensibilidade dos instrumentos. Diluições seriadas da Solução de Controle Tripsina pode ser usado para gerar as soluções controle. A leitura igual a 120% do valor obtido com a amostra em branco é considerado significativo. Rotineiramente, um limite de detecção de 5 ng de tripsina foi obtida com este procedimento. Recomenda-se que pelo menos um 5 ng controle de tripsina ser executado a cada ensaio. A solução de controle de tripsina com uma concentração de 0,5 mg / ml resultaria na desejado 5 ng de tripsina no ensaio. A diluição de 40 vezes da Solução de Controle de tripsina (20 mcg / ml) resulta em uma solução de controle de 0,5 ug / ml, ou seja, uma parte da solução de tripsina de controle para 39 partes de tampão de incubação. <img src="/files/ftp_upload/1514/figure1.jpg" alt="Figura 1" /> Figura 1 (curva padrão de atividade de tripsina): A Solução de Controle Tripsina (20 mcg / ml) também pode ser usado para gerar uma curva padrão, fazendo diluições em série (ver Figura 1). A leitura de fluorescência de cada amostra de controlo foi corrigido subtraindo-se a leitura de fluorescência da amostra em branco (FLUblank) do valor de cada amostra de controlo (FLUcontrol). Curva padrão típico de tripsina (Product Code T 6567) utilizando este kit e amostras de controle que varia de 0,15 mg / ml (1,5 ng) a 2,5 mg / ml (25 ng). Esta curva foi gerada através do procedimento descrito, com um tempo de incubação de 30 minutos para a etapa 4. Medições de fluorescência foram feitos em uma placa com vários poços. Isotiocianato de fluoresceína é fornecido como um controle de calibração de instrumentos possíveis (ver instruções do fabricante apropriado) ou determinação da faixa de linearidade do sinal 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>

O autor é um funcionário da Sigma Aldrich que produz reagentes e ferramentas utilizadas neste artigo.

Temos mostrado apenas um ensaio exemplo usando o Kit de Detecção de Protease fluorescente para detectar atividade de protease em uma amostra biológica. Este kit foi otimizado para detectar uma gama diversificada de proteases encontrados em aplicações fisiológicas. Ele é adequado para a detecção de cisteína, serina, metalo e proteases aspártico, no entanto, modificações podem ser necessários para detectar alguns proteases 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

O Kit de Detecção de Protease fluorescente fornece ready-to-use reagentes para a detecção da presença de atividade da protease. Este ensaio simples para detectar atividade de protease utiliza caseína marcado com isotiocianato de fluoresceína (FITC) como substrato. Protease atividade resulta na clivagem do substrato de caseína FITC-rotulados em fragmentos menores, que não precipitam em meio ácido. Após a incubação da amostra protease e substrato, a reação é acidificada com a adição de ácido tricloroacético (TCA). A mistura é então centrifugado com o substrato formando um pellet não digerido e as menores, fragmentos de ácido solúvel remanescente em solução. O sobrenadante é neutralizado ea fluorescência do FITC-rotulados fragmentos é medido. O procedimento descrito kit detecta o controle protease tripsina em uma concentração de aproximadamente 0,5 mcg / ml (5 ng de tripsina adicionado ao ensaio). Esta sensibilidade pode ser aumentada com um maior tempo de incubação, até 24 horas. O ensaio é realizado em tubos de microcentrífuga e procedimentos são fornecidos para a detecção de fluorescência usando tinas ou placas de vários poços.

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

O Kit de Detecção de Protease fluorescente é projetado para a medição da atividade da protease usando fluorometria. Também é adequado para a detecção de vestígios de contaminação protease. O método é baseado no hydroysis proteolítica de uma formulação de um substrato de caseína FITC-rotulados.

Uso do Kit de Detecção de Protease Fluorescente para determinar a atividade de protease

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

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JoVE Journal

Biology

28.1K visualizações.  Sigma Aldrich. O Kit de Detecção de Protease fluorescente é projetado para a medição da atividade da protease usando fluorometria. Também é adequado para a detecção de vestígios de contaminação protease. O método é baseado no hydroysis proteolítica de uma formulação de um substrato de caseína FITC-rotulados.

Vídeo: 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.

Purificação de DNA plasmidial colônias bacterianas Usando o Kit 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 ...

Biologia

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.

Imuno-histoquímica: Seções de parafina Usando o Kit de Vectastain ABC 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.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens 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="http://jove.com/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.

Não-específicas da Sigma ensaio de atividade de protease - A caseína como substrato

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.

Detecção de Ubiquitinação Protein

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.

Detecção direta da Atividade Acetato de formação do Acetato Quinase Enzima

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.

Criopreservação do mouse Sperm and Recovery utilizando o I · 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 ...

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.

Protease e catalisada por ácido Workflows Labeling Empregando<sup&gt; 18</sup&gt; O-enriquecido Água

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.

Rato Ilhéu de Langerhans isolamento usando uma combinação de colagenase purificada e Protease 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.

Determinando Membrane Topology Protein Usando Fluorescência 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 ...