Vor Beginn des Tests, müssen wir sicherstellen, dass die folgenden Reagenzien richtig vorbereitet sind: <ol><li> Eine 50 mM Kaliumphosphatpuffer, pH 7,5. Bereiten Sie mit 11,4 mg / ml Kalium Phosphat zweiwertigen Trihydrat in gereinigtem Wasser und Einstellen des pH mit 1 M HCl. Diese Lösung wird bei 37 ° C vor dem Einsatz platziert. </li><li> A 0,65% Gewicht / Volumen Casein-Lösung durch Mischen 6,5 mg / ml Casein in der 50 mM Kaliumphosphatpuffer vorbereitet. Allmählich stieg die Temperatur der Lösung unter leichtem Rühren auf 80-85 ° C für ca. 10 Minuten, bis eine homogene Dispersion erreicht ist. Es ist sehr wichtig, nicht die Lösung kochen. Der pH-Wert wird dann angepasst, wenn nötig mit NaOH und HCl. </li><li> A 110 mM Trichloressigsäure-Lösung durch Verdünnen einer 6.1N Lager 1.55 mit gereinigtem Wasser hergestellt. Trichloressigsäure ist eine starke Säure und sollten mit Vorsicht behandelt werden. </li><li> 0,5 mM Folin &amp; Ciolcaltea ist, oder Folin ist Phenolreagenz, die die Lösung, die mit Tyrosin reagieren wird, um eine messbare Farbwechsel, die direkt auf die Aktivität von Proteasen bezogen werden zu generieren. Folin ist Phenolreagenz ist eine Säure und sollte mit Sorgfalt behandelt werden. </li><li> A 500 mM Natriumcarbonatlösung, hergestellt unter Verwendung von 53 mg / ml anyhydrous Natriumcarbonat in gereinigtem Wasser. Ein Enzym, Verdünnungsmittel Lösung, die von 10 mM Natriumacetat-Puffer mit 5 mM Calcium Acetate, pH 7,5, besteht bei 37 ° C. Diese Lösung ist es, was wir feste Protease-Proben auflösen oder verdünnten Enzym-Lösungen verwenden. </li><li> 1,1 mM L-Tyrosin-Standard-Stammlösung. Hergestellt unter Verwendung von 0,2 mg / ml L-Tyrosin in gereinigtem Wasser und leicht, bis die Tyrosin löst erwärmt. Wie mit dem Casein, nicht kochen diese Lösung. Lassen Sie die L-Tyrosin-Standard auf Raumtemperatur abkühlen. Diese Lösung wird weiter verdünnt werden, um unsere Standard-Kurve machen. </li><li> Protease-Lösung. Unmittelbar vor der Anwendung aufgelöst Protease in Enzym-Verdünnungslösung in Schritt 6 erstellt. </li></ol> Wenn nötig, verwenden Sie eine feste Protease-Probe von vorbestimmten Tätigkeit, die aufgelöst mit Enzym Verdünnungsmittel auf 0,1-0,2 Einheiten / ml ist. Diese Lösung dient als positive Kontrolle für die Qualitätskontrolle Test und Validierung für die Berechnungen führen wir die Enzymaktivität bestimmt wird. Einrichten des Protease-Assay-und Standard-Kurven <ol><li> Zu Beginn dieses Tests finden Sie passende Fläschchen, dass etwa 15 mls halten wird. Für jedes Enzym, das getestet werden soll, sind 4 Flaschen benötigt. Ein Fläschchen wird als Rohling verwendet werden, und drei andere wird Assay Aktivität von drei Verdünnungen der Protease verwendet werden. Drei Verdünnungen sind nützlich, wenn die Überprüfung endgültigen Berechnungen gegeneinander. <ol><li> Zu jedem Satz von vier Flaschen, fügen 5mls unserer 0,65% Casein-Lösung. Lassen Sie sie in ein Wasserbad Gleichgewicht bei 37 ° C für ca. 5 Minuten. </li><li> Fügen unterschiedlicher Menge Enzymlösung, dass drei der Probe Fläschchen getestet werden, aber nicht die leer. Mix durch Schütteln und Inkubation für 37 ° C für genau 10 Minuten. Die Protease-Aktivität und Folgeschäden Befreiung von Tyrosin während dieser Inkubationszeit ist, was wird gemessen und verglichen werden zwischen Testproben. </li></ol></li><li> Nach diesem 10-minütigen Inkubation, fügen Sie die 5 ml des TCA-Reagenz in jedes Röhrchen, um die Reaktion zu stoppen. Dann fügen Sie ein geeignetes Volumen der Enzymlösung in jedes Röhrchen, auch die leeren, so dass die endgültige Volumen der Enzymlösung in jedes Röhrchen wird 1 ml. Dies ist für den Extinktionswert des Enzyms selbst Rechnung und um sicherzustellen, dass das Endvolumen in jeder Röhre gleich ist getan. Inkubieren Sie die Lösungen bei 37 ° C für 30 Minuten. Während dieser 30-minütigen Inkubation, können Sie zum Einrichten Ihres Tyrosin Standardverdünnungen. Verwende 6 dram Fläschchen (dram Fläschchen mit Polypropylen-Röhrchen ersetzt werden), die leicht zu halten kann 8 mls. Um die sechs Fläschchen, fügen Sie die 1,1 mM Tyrosin Standard-Stammlösungen mit folgenden Volumina in ml: 0,05, 0,10, 0,20, 0,40, 0,50. Fügen Sie keine Tyrosin-Standard auf den Rohling. Niedrigere Standards können für unreine Proben, die wenig Farbe ändern Ertrag benötigt werden. Sobald die Tyrosin-Standardlösung zugesetzt worden ist, fügen Sie ein geeignetes Volumen des gereinigten Wassers in jedem der Standards, um die Lautstärke zu 2 ml zu bringen. </li><li> Nach der Inkubationszeit von 30 Minuten, Filter jede der Testlösungen und der Rohling mit einem 0,45 um Polyethersulfon Spritzenfilter. Filtration ist erforderlich, um unlösliche Bestandteile aus den Proben zu entfernen. <ol><li> Fügen Sie die Filtration 2 ml der Test-und Blindproben Filtrat bis 4 dram Fläschchen, dass mindestens 8 mls halten kann. Die gleiche Art von Flasche, in der die Standards hergestellt wurden genutzt werden kann. </li><li> Um alle Flaschen mit den Normen und Standards blank, fügen 5mls von Natriumcarbonat. Für beste Ergebnisse 1 ml Folin-Reagenz unmittelbar danach. </li><li> Add Natriumcarbonat zu einer pH-Abfall durch die Zugabe des Folin-Reagenz geschaffen zu regulieren. </li><li> Add Natriumcarbonat zu den Proben und test leer. Diese Lösungen trüb nach der Zugabe von Natriumcarbonat. Fügen Sie die Folin-Reagenz, das vor allem reagiert mit freiem Tyrosin. </li><li> Mischen Sie die DRAM-Fläschchen durch Schütteln und Inkubation bei 37 ° C für 30 Minuten. </li></ol> Nach dieser Inkubation, sollten Sie feststellen, dass die Standards eine Abstufung der Farbe korreliert mit der Menge an Tyrosin hinzugefügt haben, die höchsten Konzentrationen von Tyrosin erscheinen dunkelsten. Sie können auch feststellen, nennenswerte Farbveränderung in unseren Proben. 2mls dieser Lösungen sind durch ein 0,45 um Polyethersulfon Spritzenfilter in geeignete Küvetten. Nun, da der Test durchgeführt wird, können Sie mit dem Spektralfotometer Sie diese in unserem Extinktionswerte aufzunehmen. </li></ol> Messung der Absorption und Berechnung Enzymaktivität <ol><li> Die Absorption der Proben wird durch ein Spektralphotometer mit einer Wellenlänge von 660nm gemessen. Der Lichtweg ist 1cm gesetzt. Notieren Sie sich die Extinktionswerte für die Standards, Standard-blank, die verschiedenen Proben und testen leer. Sobald alle Daten gesammelt wurden, kann die Standardkurve erstellt werden. Um die Kurve zu erzeugen, Extinktionsdifferenz zwischen der Standard-und Standard-Rohling berechnet werden muss. Dies ist der Extinktionswert auf die Menge an Tyrosin in der Standard-Lösungen. Nach dieser einfachen Rechnung, erstellen Sie die Standard-Kurve mit einem Grafik-Programm, um die Änderung der Absorption von unseren Standards auf der Y-Achse gegen die Menge in Mikromol für jedes unserer 5-Norm auf der X-Achse zeichnen. <table style="width: 416px; height: 118px;" border="0"><tbody><tr><th style="text-align:center"> Volume von Tyrosin Standard- </th><th style="text-align:center"> uMoles Tyrosin </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> Nachdem die Daten eingegeben sind, erzeugen eine Reihe von Best-Fit und entsprechende Neigung Gleichung. Finden Sie die Änderung der Absorption in den Proben durch Berechnung der Differenz zwischen der Probe Absorption und der Absorption der Test leer. Einsetzen der Extinktionswert für eine der Proben in den Hang Gleichung ein und lösen in der Mikromol Tyrosin befreiten während dieser besonderen proteolytischen Reaktion führen. Um die Aktivität des Enzyms in Einheiten pro / ml, führen Sie die folgende Berechnung: (Umole Tyrosinäquivalent freigegeben) x (11) Einheiten / ml Enzym = __________________________________________ (1) x (10) x (2) 11 = Gesamtvolumen (in Milliliter)-Assay 10 = Time of Assay (in Minuten) nach der Einheit Definition 1 = Volumen der Enzyme (in Milliliter) des Enzyms verwendet 2 = Volumen (ml) in kolorimetrischen Bestimmung verwendet Nehmen Sie die Anzahl der Mikromol Tyrosin-Äquivalente freigesetzt aus der Steigung Gleichung erhalten und vermehren es durch das Gesamtvolumen des Assays in mls, was in unserem Fall ist 11mls. Teilen Sie diesen Wert durch drei weitere Mengen: die Zeit des Tests, die wir für 10 Minuten lief, das Volumen des Enzyms im Assay verwendet, die variiert wurde (wir verwenden 1ml), das Volumen der Milliliter in kolorimetrischen Nachweis verwendet, der eventuell unterscheiden auf der Grundlage Ihrer Küvette. Wir haben 2 mls. </li><li> Mikromol Tyrosin durch die Zeit in Minuten liefert die Messung der Protease-Aktivität als &quot;Einheiten&quot; unterteilt. Wir können heben die Einheiten für Volumen-Messung in den Zähler und Nenner, so dass ein Messen von Enzymaktivität in Einheiten / ml. Um die Aktivität in einem festen Protease Probe in Enzym-Verdünnungsmittel verdünnt bestimmen, teilen wir unsere Tätigkeit in Einheiten / ml durch die Konzentration der festen in diesem Test ursprünglich in mg / ml. Verwendet, so dass uns mit der Aktivität in Bezug auf die Einheiten / mg . Einheiten / ml Enzym Units / mg Feststoff = _____________________ mg Feststoff / ml Enzym </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>

Wir haben gerade gezeigt, wie Sie Protease-Aktivität analysieren mit Hilfe von Sigma universelle Protease-Aktivität-Assay. Darüber hinaus ist dieser Test nützlich, um sicherzustellen, dass unsere Proteasen haben genau Aktivität bestimmt, bevor Sie sie erhalten für Ihre Experimente. Wie Sie gesehen haben, wenn Sie diese Prozedur, ist es von größter Wichtigkeit zu erinnern, um sowohl das Kasein und Tyrosin-Lösungen Wärme langsam und nicht zu kochen. Außerdem ist es wichtig, verschiedene Rohlinge für Ihre Ansprüche und für jede Probe, die Sie vorbereiten.

<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

Proteasen bauen Peptidbindungen. Im Labor ist es oft notwendig, um zu messen und / oder vergleichen Sie die Aktivität von Proteasen. Sigma ist nicht-spezifische Protease-Aktivität Test kann als ein standardisiertes Verfahren eingesetzt werden, um die Aktivität der Proteasen, die das, was wir während unserer Qualitätskontrolle zu tun ist zu bestimmen. In diesem Test wirkt Casein als Substrat. Wenn die Protease testen wir Casein verdaut, ist die Aminosäure Tyrosin befreit zusammen mit anderen Aminosäuren-und Peptid-Fragmente. Folin und Ciocalteus Phenol oder Folin-Reagenz in erster Linie reagiert mit freiem Tyrosin zu einer blau gefärbten Chromophor, die quantifizierbar und gemessen als OD-Wert auf dem Spektralphotometer ist zu produzieren. Je mehr Tyrosin, die aus Kasein freigesetzt wird, desto mehr Chromophore erzeugt und desto stärker ist die Aktivität der Protease. Extinktionswerte durch die Aktivität der Protease erzeugt werden, um eine Standardkurve, die durch Umsetzung von bekannten Mengen von Tyrosin mit dem FC-Reagenz auf Veränderungen in der Absorption mit der Menge an Tyrosin in Mikromol korrelieren erzeugt wird verglichen. Von der Standardkurve der Aktivität der Protease Proben können in Bezug auf die Einheiten, die über den Betrag in Mikromol Tyrosin-Äquivalente aus Kasein pro Minute freigesetzt bestimmt werden. Um diesen Artikel in Chinesisch, <a href="http://jove.com/index/details.stp?ID=910">klicken Sie hier</a>

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

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

Proteasen bauen Peptidbindungen. Im Labor ist es oft notwendig, um zu messen und / oder vergleichen Sie die Aktivität von Proteasen. Sigma ist nicht-spezifische Protease-Aktivität Test kann als ein standardisiertes Verfahren eingesetzt werden, um die Aktivität von Proteasen zu bestimmen.

Sigma Unspezifische Protease Activity Assay - Casein als Substrat

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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Serie: 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.

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.

Schnelle Genotypisierung von Maus Tissue Mit Sigma 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 ...

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Biologie

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.

Die Nutzung der Protease Fluorescent Detection Kit für Protease-Aktivität zu bestimmen

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.

Ein Assay zur Messung der Aktivität von Escherichia coli Inducible Lysin 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.

Ein Matrigel-Based Rohr Formation Assay zur vaskulären Aktivität von Tumorzellen Beurteilen

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

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Untersuchen der Kinase-Aktivität von LRRK2 In-vitro-</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

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-und Acid-katalysierten Labeling Workflows beschäftigt<sup&gt; 18</sup&gt; O-angereichertem Wasser

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.

Maus Islet der Langerhans Isolation durch eine Kombination von gereinigtem Collagenase und 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 ...

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.

Bestimmen Membrane Protein Topologie Mit Fluorescence Protease Schutz (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 als ein indirektes Maß für Systemische Oxidativer Stress

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.

Identifikation von Kinase-Substrat-Pairs Mit 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 ...