Prima di iniziare il test, dobbiamo fare in modo che i seguenti reagenti sono correttamente preparati: <ol><li> A 50 mM tampone fosfato di potassio, pH 7,5. Preparare con 11,4 mg / ml di fosfato di potassio bibasico, triidrato in acqua purificata e la regolazione del pH con HCl 1M. Questa soluzione è posizionato a 37 ° C prima dell&#39;uso. </li><li> Un 0,65% peso / volume soluzione di caseina, preparato miscelando 6,5 mg / ml di caseina nel buffer 50 mM fosfato di potassio. Gradualmente aumentato la temperatura della soluzione con delicata agitazione a 80-85 ° C per circa 10 minuti fino a una dispersione omogenea è raggiunto. E &#39;molto importante non far bollire la soluzione. Il pH è quindi regolata, se necessario, con NaOH e HCl. </li><li> A 110 mM soluzione di acido tricloroacetico, preparata diluendo un magazzino 1:55 6.1N con acqua purificata. L&#39;acido tricloroacetico è un acido forte e deve essere maneggiato con cura. </li><li> 0.5 mM Folin &amp; Ciolcaltea, o reagente fenolo Folin, che è la soluzione che reagisce con tirosina per generare un cambiamento misurabile colore che saranno direttamente collegati con l&#39;attività delle proteasi. Reagente Folin fenolo è un acido e deve essere maneggiato con cura. </li><li> A mm 500 carbonato di sodio soluzione, preparata con 53 mg / ml di carbonato di sodio anyhydrous in acqua purificata. Una soluzione diluente enzima, che è composto da 10 mM tampone di acetato di sodio con calcio acetato 5mm, pH 7,5, a 37 ° C. Questa soluzione è quello che usiamo per sciogliere i campioni della proteasi solidi o soluzioni diluite enzima. </li><li> 1,1 mm L-tirosina soluzione madre standard. Preparati con 0,2 mg / ml L-tirosina in acqua depurata e riscaldata dolcemente fino alla tirosina si dissolve. Come con la caseina, non bollire questa soluzione. Lasciare che la L-tirosina standard per raffreddare a temperatura ambiente. Questa soluzione sarà diluita ulteriormente per rendere la nostra curva standard. </li><li> Proteasi soluzione. Immediatamente prima dell&#39;uso, sciogliere proteasi in soluzione diluente enzimatica preparata al punto 6. </li></ol> Se necessario, utilizzare un campione solido proteasi di attività predeterminate, che viene disciolto utilizzando un diluente enzima di 0,1-0,2 unità / ml. Questa soluzione serve come controllo positivo per il test di controllo qualità e la convalida per i calcoli che si esibiranno per determinare l&#39;attività enzimatica. Impostazione del saggio della proteasi e Curve standard <ol><li> Per iniziare questo saggio, trovano fiale adatto che conterrà circa 15 ml. Per ogni enzima che sarà testato, 4 flaconcini sono necessari. Un flaconcino sarà usato come un vuoto, e tre altri saranno utilizzati per attività di analisi di tre diluizioni della proteasi. Tre diluizioni sono utili per la verifica dei calcoli finale contro l&#39;altro. <ol><li> Per ogni serie di quattro fiale, aggiungere 5mls della nostra soluzione di caseina 0,65%. Lasciate che equilibrano in un bagno d&#39;acqua a 37 ° C per circa 5 minuti. </li><li> Aggiungi variando volumi di soluzione enzimatica che sarà testato per tre fiale campione del test, ma non il vuoto. Mescolare agitando e incubare per 37 ° C per dieci minuti esatti. L&#39;attività della proteasi e liberazione consequenziali di tirosina durante questo periodo di incubazione è quello che sarà misurato e confrontato tra i campioni di prova. </li></ol></li><li> Dopo questa incubazione di 10 minuti, aggiungere i 5 ml di reagente TCA a ciascuna provetta per arrestare la reazione. Quindi, aggiungere un volume adeguato di soluzione enzimatica a ciascuna provetta, anche il vuoto, in modo che il volume finale della soluzione enzimatica in ogni tubo è di 1 ml. Questo viene fatto per conto del valore di assorbanza dello stesso enzima e per garantire che il volume finale di ogni tubo è uguale. Incubare le soluzioni a 37 ° C per 30 minuti. Durante questa incubazione di 30 minuti, si consiglia di impostare il diluizioni tirosina standard. Usa 6 flaconi dram (fiale DRAM possono essere sostituiti con tubi in polipropilene) che può facilmente contenere 8 ml. Per i sei fiale, aggiungere il 1,1 mm soluzioni tirosina magazzino di serie con i seguenti volumi in ml: 0,05, 0,10, 0,20, 0,40, 0,50. Non aggiungere alcun tipo di tirosina al vuoto. Abbassare gli standard possono essere necessari per campioni impuri che darà piccolo cambiamento di colore. Una volta che la soluzione di tirosina standard è stato aggiunto, aggiungere un volume adeguato di acqua depurata a ciascuno degli standard per portare il volume a 2 ml. </li><li> Dopo l&#39;incubazione di 30 minuti, filtrare ciascuna delle soluzioni di test e il vuoto usando una siringa da 0,45 um polietersulfone filtro. La filtrazione è necessario per rimuovere eventuale insoluto dai campioni. <ol><li> Aggiungere la filtrazione 2 mls dei campioni di prova e nel bianco filtrato a 4 flaconcini DRAM che può contenere almeno 8 ml. Lo stesso tipo di fiala in cui sono stati preparati gli standard può essere utilizzato. </li><li> Per tutti i flaconi contenenti gli standard e vuoto standard, aggiungere 5mls di carbonato di sodio. Per ottenere i migliori risultati, aggiungere 1 ml di reattivo di Folin immediatamente dopo. </li><li> Aggiungi carbonato di sodio per regolare ogni goccia di pH creato con l&#39;aggiunta di reagente di Folin ha. </li><li> Aggiungi carbonato di sodio per i campioni di prova e test vuoto. Queste soluzioni diventa opaco dopo l&#39;aggiunta di carbonato di sodio. Aggiungere il reagente di Folin, che reagiscono soprattutto con tirosina libero. </li><li> Mescolare le fiale dram agitando e incubare a 37 ° C per 30 minuti. </li></ol> Dopo questa incubazione, si dovrebbe notare che le norme hanno una gradazione di colore correlazione con la quantità di tirosina aggiunta: la più alta concentrazione di tirosina che appaiono più scure. Si può notare anche modificare sensibilmente il colore nei nostri campioni di prova. 2mls di queste soluzioni sono filtrate con un 0,45 polietersulfone siringa filtro in cuvette adatto. Ora che il test viene eseguito, si può procedere allo spettrofotometro per registrare i nostri valori di assorbanza. </li></ol> Misurare l&#39;assorbanza e calcolo attività enzimatica <ol><li> L&#39;assorbanza dei campioni si misura con uno spettrofotometro utilizzando una lunghezza d&#39;onda di 660 nm. Il percorso della luce è impostato su 1 cm. Registrare i valori di estinzione di tali norme, vuoto standard, i campioni di prova diversi, e prova in bianco. Una volta che tutti i dati sono stati raccolti, la curva standard può essere creato. Al fine di generare la curva, la differenza di assorbanza tra il bianco standard e standard devono essere calcolati. Questo è il valore di assorbanza attribuibile alla quantità di tirosina nelle soluzioni standard. Dopo questo semplice calcolo, creare la curva standard utilizzando un programma grafico per tracciare la variazione di assorbanza dei nostri standard sull&#39;asse Y, contro l&#39;importo in micromoli per ciascuno dei nostri 5 standard sull&#39;asse X. <table style="width: 416px; height: 118px;" border="0"><tbody><tr><th style="text-align:center"> Volume standard di tirosina </th><th style="text-align:center"> uMoles Tirosina </th></tr><tr><td style="text-align:center"> 0,05 </td><td style="text-align:center"> 0,055 </td></tr><tr><td style="text-align:center"> 0,10 </td><td style="text-align:center"> 0,111 </td></tr><tr><td style="text-align:center"> 0,20 </td><td style="text-align:center"> 0,221 </td></tr><tr><td style="text-align:center"> 0,40 </td><td style="text-align:center"> 0,442 </td></tr><tr><td style="text-align:center"> 0,50 </td><td style="text-align:center"> 0,553 </td></tr></tbody></table></li><li> Dopo i punti dati sono stati inseriti, generare una retta di regressione e l&#39;equazione pendenza corrispondente. Trovare la variazione di assorbanza nei campioni analizzati calcolando la differenza tra l&#39;assorbanza campione da analizzare e l&#39;assorbanza del bianco prova. Inserimento del valore di assorbanza per uno dei campioni di prova nell&#39;equazione pendenza e risolvere comporterà la micromoli di tirosina liberato durante questa reazione proteolitica particolare. Per ottenere l&#39;attività dell&#39;enzima in unità per / ml, eseguire il seguente calcolo: (Equivalenti tirosina umole rilasciato) x (11) Unità / ml = enzima __________________________________________ (1) x (10) x (2) 11 = il volume totale (in millilitri) di test 10 = Tempo di test (in minuti) secondo la definizione di unità 1 = Volume di enzimi (in millilitri) di enzima usato 2 = volume (in millilitri) utilizzati nella determinazione colorimetrica Prenda il numero di equivalenti micromoli tirosina rilasciato ottenuto dall&#39;equazione pendenza e moltiplicarlo per il volume totale del test in MLS, che nel nostro caso è 11mls. Dividere questo valore per tre quantità di altri: il tempo del test, che abbiamo corso per 10 minuti, il volume di enzima utilizzato nel test, che è stato variato (usiamo 1ml), il volume di millilitri utilizzati nel rilevamento colorimetrico, che può differiscono in base alla cuvetta. Abbiamo usato 2 ml. </li><li> Micromoli di tirosina divise da tempo nella misurazione minuto i rendimenti di attività della proteasi chiamata &quot;unità&quot;. Siamo in grado di annullare le unità di misura del volume nel numeratore e denominatore, lasciando un measurment di attività enzimatica in termini di unità / ml. Al fine di determinare l&#39;attività in un campione della proteasi solida diluito in diluente enzima, dividiamo la nostra attività in unità / ml per la concentrazione di solidi utilizzati in questo saggio originariamente in mg / ml., Lasciandoci con l&#39;attività in termini di unità / mg . Unità / ml di enzima Unità / mg solido = _____________________ mg solidi / ml di enzima </li></ol>

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

Abbiamo appena mostrato come analizzare l&#39;attività della proteasi con Sigma universale saggio di attività della proteasi. Inoltre, questo test è utile per garantire che i nostri proteasi hanno determinato con precisione l&#39;attività prima di ricevere per i vostri esperimenti. Come avete visto, durante questa procedura, è di fondamentale importanza per ricordare al calore, sia la caseina e le soluzioni tirosina lentamente e non a bollire loro. Inoltre, è fondamentale per preparare diversi spazi sia per i vostri standard e per ogni campione che si ha.

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

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

Proteasi rompere i legami peptidici. Nel laboratorio, è spesso necessario misurare e / o confrontare l&#39;attività della proteasi. Sigma non-specifico saggio di attività della proteasi può essere utilizzato come una procedura standardizzata per determinare l&#39;attività della proteasi, che è quello che facciamo durante le procedure di qualità del nostro controllo. In questo saggio, la caseina agisce come substrato. Quando la proteasi stiamo testando digerisce caseina, la tirosina viene liberato insieme ad altri aminoacidi e frammenti peptidici. Reagente Folin e Ciocalteus fenolo, o Folin reagisce principalmente con tirosina liberi di produrre un cromoforo di colore blu, che è quantificabile e misurato come valore in assorbanza allo spettrofotometro. La tirosina più che viene rilasciato dalla caseina, più i cromofori sono generati e più forte è l&#39;attività della proteasi. Valori di assorbanza generata dalla attività della proteasi vengono confrontati con una curva standard, che viene generato dalla reazione quantità note di tirosina con il reagente FC di correlare i cambiamenti di assorbanza con la quantità di tirosina in micromoli. Dalla curva standard dell&#39;attività di campioni della proteasi possono essere determinati in termini di unità, che è la quantità in micromoli di equivalenti tirosina rilasciato da caseina al minuto. Per visualizzare questo articolo in cinese, <a href="http://jove.com/index/details.stp?ID=910">clicca qui</a>

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

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

Proteasi rompere i legami peptidici. Nel laboratorio, è spesso necessario misurare e / o confrontare l&#39;attività della proteasi. Sigma non-specifico saggio di attività della proteasi può essere utilizzato come una procedura standardizzata per determinare l&#39;attività della proteasi.

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 ...

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

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

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

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

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

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

Genotipizzazione rapida del tessuto mouse utilizzando Sigma Extract-N-Amp PCR Tissue Kit

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

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Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

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 ...

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

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

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

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

Un test per misurare l&#39;attività di Escherichia coli Inducibile Lisina Decarboxyase

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

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

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

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

Un Matrigel-Based Formazione Assay tubo per valutare l&#39;attività vascolare delle cellule tumorali

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

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

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

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

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

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 ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

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

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

Comet Assay come una misura indiretta della sistemica Stress ossidativo

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

Identification of Kinase-substrate Pairs Using High Throughput Screening

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

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

Identificazione delle coppie Kinase-substrato Usando High Throughput Screening


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

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

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

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

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