Istruzioni per la preparazione Il tampone di incubazione, tampone del saggio, e 0,6 N Soluzione TCA sono forniti pronti per l&#39;uso-solutions. La FITC-caseina substrato - Si consiglia di aliquota con FITC caseina substrato in volumi più piccoli al loro arrivo al fine di evitare ripetuti cicli di gelo-disgelo. Se il FITC-caseina substrato è sottoposta a ripetuti cicli di gelo e disgelo, un leggero aumento del fondo avverrà, quindi, abbassando la sensibilità. Le aliquote devono essere conservati a -20 ° C e al riparo dalla luce. Ogni campione, reazione vuoto, o di controllo richiede 20 microlitri della FITC-caseina substrato. Gelo / disgelo e vigorosa miscelazione o agitazione può causare la FITC a separarsi dal caseina con conseguente maggiore sfondo lettura e causando meno substrato ad essere disponibile per la scissione della proteasi. L&#39;isotiocianato di fluoresceina (FITC) Soluzione di controllo-Il controllo FITC può essere ricostituito nel tampone del saggio alla concentrazione adeguata. Questa soluzione deve essere fresco e al riparo dalla luce. Soluzione finale della tripsina di controllo - Aggiungere 100 ml di 1 mM HCl al flaconcino di tripsina, proteasi Control (Product Code T 6567). Miscelare brevemente per garantire la tripsina è sciolto. Aggiungere 900 ml di tampone di incubazione e mescolare bene. In alternativa, i buffer possono essere utilizzati altri se lo si desidera per il test. La concentrazione finale di lavoro della tripsina è di 20 mg / mL. Quando si è pronti per preparare la soluzione di controllo tripsina, combinano una aliquota della soluzione acida tripsina con la giusta quantità di Incubation Buffer (1 tripsina acidificato parte a 9 tampone parti). Memorizzazione della tripsina in condizioni acide aumenta la stabilità della tripsina. La soluzione di controllo tripsina è sensibile alla temperatura e non è stabile a basse concentrazioni. Si prega di rivedere bollettino tecnico del kit per la conservazione e la stabilità di queste soluzioni preparate. Procedura <ol><li> È più facile preparare i controlli, spazi vuoti, e tutti i campioni in una sola volta. Per ogni campione, aggiungere 20 ml di tampone di incubazione, 20 l di FITC-caseina substrato, e 10 ml di campione in una provetta da microcentrifuga. Per i campioni di prova con l&#39;attività della proteasi alta diluizione del campione può essere richiesto. </li><li> Preparare i campioni di controllo appropriate (vedere Esempi di controllo) con l&#39;aggiunta di 20 l di tampone di incubazione, 20 l di FITC-caseina substrato, e 10 ml di campione di controllo di una provetta. </li><li> Preparare un campione in bianco con l&#39;aggiunta di 20 l di tampone di incubazione, 20 l di FITC-caseina substrato, e 10 ml di acqua ultrapura in una provetta da microcentrifuga. </li><li> Mescolare delicatamente ogni tubo ed incubare a 37 ° C al buio per 60 minuti. Fare attenzione a non mescolare troppo energicamente, come turbolenza eccessiva può causare alta fluorescenza di fondo e di ridurre la sensibilità del test. Nota: il tempo di incubazione può essere prolungato fino a 24 ore per aumentare la sensibilità. Fare attenzione a non superare 24 ore come FITC-caseina può cominciare a degradare, portando ad alta fluorescenza di fondo. </li><li> Dopo l&#39;incubazione aggiungere 150 microlitri della soluzione 0,6 N di acido tricloroacetico ad ogni provetta. TCA è molto corrosivo e deve essere maneggiato mentre indossa l&#39;equipaggiamento protettivo idoneo. </li><li> Mescolare delicatamente e incubare a 37 ° C al buio per 30 minuti. </li><li> Centrifugare le provette per 10 minuti a 10.000 x g. Il supernatante contiene il solubile acido, frammenti marcato con FITC ed è utilizzato per la misurazione della fluorescenza. </li></ol> Misure di fluorescenza Questi metodi possono essere scalato verso l&#39;alto o verso il basso a seconda delle esigenze della strumentazione disponibile. Per il confronto con una curva standard preparata con i campioni di controllo adeguati, sottrarre la lettura di fluorescenza del campione in bianco (FLUblank) dal valore di ciascun campione (FLUtest). Cuvette <ol><li> Pipettare 10 ml di surnatante (passo 7) e 1 ml di tampone del saggio nella cuvetta adatto e mescolare delicatamente. Nota: la soluzione del Buffer surnatante e di analisi possono essere conservati al buio a 2-8 ° C fino a 24 ore prima di misurare la fluorescenza. </li><li> Registrare l&#39;intensità di fluorescenza con eccitazione a 485 nm e il monitoraggio della lunghezza d&#39;onda di emissione di 535 nm. </li></ol> Pozzetti Piastre <ol><li> Pipettare 10 ml di nota surnatante (punto 7) e 1 ml di tampone del saggio in un tubo adatto o fiala e mescolare delicatamente: la soluzione del Buffer surnatante e di analisi possono essere conservati al buio a 2-8 ° C al massimo a 24 ore prima di misurare la fluorescenza. </li><li> Trasferire 200 ul di un pozzo nero di una piastra da 96 pozzetti. Registrare l&#39;intensità di fluorescenza con eccitazione a 485 nm e il monitoraggio della lunghezza d&#39;onda di emissione di 535 nm. O </li></ol><ol><li> Pipettare 2 ml di surnatante (passo 7) e 200 microlitri della B AssayUffer in un pozzo nero di una piastra da 96 pozzetti. Nota: la soluzione del Buffer surnatante e di analisi possono essere conservati al buio a 2-8 ° C fino a 24 ore prima di misurare la fluorescenza. </li><li> Registrare l&#39;intensità di fluorescenza con eccitazione a 485 nm e il monitoraggio della lunghezza d&#39;onda di emissione di 535 nm. </li></ol> Campioni di controllo La soluzione di controllo Tripsina può essere utilizzato per confermare il test funziona correttamente, per determinare il limite di rilevazione, o creare una curva generale standard. Per l&#39;analisi di un diverso, specifico della proteasi, si consiglia di preparare una soluzione di controllo contenente le proteasi specifica nel buffer di incubazione appropriato. Il limite di rilevazione del test è la quantità di proteasi che produce una lettura significativa fluorescenza al di sopra del valore ottenuto con il campione bianco. Il limite di rilevazione varia a seconda della sensibilità della strumentazione. Diluizioni seriali della soluzione di controllo Tripsina possono essere utilizzati per generare le soluzioni di controllo. Una lettura pari al 120% del valore ottenuto con il campione in bianco è considerato significativo. Di routine, un limite di rilevazione di 5 ng di tripsina è stato ottenuto con questa procedura. Si raccomanda che almeno un 5 ng controllo tripsina essere eseguita per ogni dosaggio. Una soluzione di controllo di tripsina con una concentrazione di 0,5 mg / ml porterebbe alla desiderato 5 ng di tripsina nel saggio. A 40 volte la diluizione della soluzione di controllo tripsina (20 mg / ml) si traduce in un mcg / 0,5 ml soluzione di controllo, cioè una parte della soluzione di controllo tripsina a 39 parti di tampone di incubazione. <img src="/files/ftp_upload/1514/figure1.jpg" alt="Figura 1" /> Figura 1 (curva standard di attività tripsina): La soluzione di controllo tripsina (20 mg / ml) può anche essere usato per generare una curva standard, rendendo diluizioni seriali (vedi Figura 1). La lettura della fluorescenza di ciascun campione di controllo è stato corretto sottraendo la lettura di fluorescenza del campione in bianco (FLUblank) dal valore di ciascun campione di controllo (FLUcontrol). Tipica curva standard per la tripsina (Codice prodotto T 6567) utilizzando questo kit e campioni di controllo che vanno da 0,15 mg / ml (1,5 ng) a 2,5 mg / ml (25 ng). Questa curva è stata generata seguendo la procedura descritta, con un tempo di incubazione di 30 minuti per il punto 4. Misure di fluorescenza sono state effettuate in un piatto pozzetti. Isotiocianato di fluorescina viene fornito come un controllo per la calibrazione dello strumento possibile (vedere le istruzioni del produttore appropriato) o la determinazione del range di linearità del segnale 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>

L&#39;autore è un dipendente della Sigma Aldrich che produce reagenti e strumenti utilizzati in questo articolo.

Abbiamo appena mostrato un saggio esempio utilizzando la fluorescenza Detection Kit della proteasi per rilevare l&#39;attività della proteasi in un campione biologico. Questo kit è stato ottimizzato per rilevare una vasta gamma di proteasi presenti nelle applicazioni fisiologiche. È adatto per il rilevamento di serina, cisteina, metallo, e aspartico proteasi, tuttavia, le modifiche possono essere tenuti a rilevare alcuni proteasi specifiche.

<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

Il fluorescente Detection Kit proteasi fornisce pronti per l&#39;uso-reagenti per la rilevazione della presenza di attività della proteasi. Questo semplice test per rilevare l&#39;attività della proteasi utilizza la caseina marcato con isotiocianato di fluorescina (FITC) come substrato. L&#39;attività della proteasi risultati nella scissione del substrato caseina coniugati con fluoresceina in frammenti più piccoli, che non precipitare in condizioni di acidità. Dopo l&#39;incubazione del campione proteasi e substrato, la reazione è acidificato con l&#39;aggiunta di acido tricloroacetico (TCA). La miscela viene poi centrifugato con il substrato digerito formando una pallina e la più piccola, frammenti di acido solubile rimane in soluzione. Il supernatante viene neutralizzata e la fluorescenza della marcata con FITC frammenti viene misurato. La procedura descritta kit rileva il controllo della proteasi tripsina ad una concentrazione di circa 0,5 mg / ml (5 ng di tripsina aggiunto al test). Questa sensibilità può essere aumentata con un tempo di incubazione più lungo, fino a 24 ore. Il test viene eseguito in provette da microcentrifuga e le procedure sono previste per la rilevazione di fluorescenza utilizzando cuvette o piastre a pozzetti multipli.

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

Il fluorescente Detection Kit proteasi è progettato per la misurazione dell&#39;attività della proteasi con fluorometria. E &#39;anche adatto per il rilevamento di tracce di contaminazione della proteasi. Il metodo si basa sulla hydroysis proteolitica di una formulazione proprietaria di un marcato con FITC substrato di caseina.

L&#39;uso del kit di rilevamento a fluorescenza della proteasi per determinare l&#39;attività della proteasi

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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Nanyang Technological University

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JoVE Journal

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28.1K Views.  Sigma Aldrich. Il fluorescente Detection Kit proteasi è progettato per la misurazione dell'attività della proteasi con fluorometria. E 'anche adatto per il rilevamento di tracce di contaminazione della proteasi. Il metodo si basa sulla hydroysis proteolitica di una formulazione proprietaria di un marcato con FITC substrato di caseina.

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.

Purificazione del DNA plasmidi da colonie batteriche Utilizzando il 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 ...

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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.

Immunoistochimica: sezioni di paraffina Utilizzando il Vectastain ABC Kit Labs Vector

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.

Etichettatura Cellule staminali con le tinture fluorescenti per la rilevazione non invasiva con 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 ...

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.

Sigma non specifiche attività di analisi della proteasi - caseina come 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.

Rilevamento di proteine ​​Ubiquitinazione

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.

Rilevazione diretta della acetato di formazione Attività di acetato enzima chinasi

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.

Crioconservazione del mouse Sperm and Recovery utilizzando il Kit · I 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.

Flussi di lavoro di etichettatura della proteasi e catalizzata da acidi Impiegando<sup&gt; 18</sup&gt; O arricchita di acqua

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.

Mouse Isolotto di Langerhans isolamento utilizzando una combinazione di Collagenasi depurato e proteasi 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.

Determinazione Membrane Protein Topologia Usando fluorescenza 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 ...