Ringraziamo il Fondo UAMS Proteomics supporto spettrometria di massa. Il finanziamento per questo progetto è stato fornito da sovvenzioni NIH P20RR015569, P20RR016460 e R01DA025755.

Questo protocollo è stato modificato da Blair et al. 6. 1. LSD1 demetilazione Assay <ol><li> In un volume finale di 20 pl, combinare 125 ng LSD1 ricombinante con 0,25 mg di metil-istone H3 peptide (ARTKme2QTARKSTGGKAPRKQLYK-biotina) in tampone demetilasi (50 mM Tris-Cl pH 8,5, 50 mM KCl, 5 mM MgCl 2, 5% glicerolo). Inoltre, eseguire un controllo solo peptide senza enzima. Il controllo è per la sezione 3 e non necessita di metilazione riduttiva. </li><li> Incubare per 2 ore a 37 ° C. </li><li> Per raccogliere i peptidi, aggiungere 8 microlitri Poros R2 sfere 20 micron a ciascun campione e agitare per 15 minuti a temperatura ambiente. Preparare perline Poros sospendendo 1 volume di perline con 1 volume di metanolo, e quindi aggiungere 10 volumi di 5% di acido formico / 0,2% TFA. </li><li> Condizione due C 18 ZipTips aspirando 20 microlitri di 0.1% TFA nella punta e spingendolo indietro con una pipetta. Fate questo due volte con lo 0,1% TFA, quattro volte con il 70% acetonitrile / 0,1% TFA, e quattro volte di nuovo con 0,1% TFA. </li><li> Caricare i due campioni (controllo e la reazione demetilasi) sulle condizionata C 18 ZipTips pipettando l&#39;impasto tallone dietro la resina C 18 nelle ZipTips e spingendo il volume attraverso con una pipetta. </li><li> Lavare ZipTips due volte con 20 pl di 0,1% TFA. </li><li> Eluire in 40 pl di 70% acetonitrile / 0,1% TFA. </li><li> Liofilizzato ogni eluente completamente con un concentratore SpeedVac. </li></ol> 2. Metilazione riduttiva Questa porzione del protocollo viene modificato da Blair et al. E Rayment et al. 6,7. <ol><li> Risospendere la reazione demetilasi in 100 pl di tampone fosfato 50 mM, pH 7,4. </li><li> Per la reazione demetilasi, aggiungere 8 pl di 15 mg / mL borano dimetilammina. Borano dimetilammina viene sciolto in tampone fosfato 50 mM, pH 7,4, per dare 15 mg / ml di soluzione concentrata.Questo dovrebbe essere fresco. </li><li> Per la reazione demetilasi, aggiungere 16 pl di 250 mM formaldeide deuterato. La 250 formaldeide mM deuterato si prepara diluendo lo stock in H 2 0. Questo dovrebbe essere fresco. </li><li> Incubare a 4 ° C per 2 ore. </li><li> Ripetere i punti 2.2, 2.3 e 2.4. </li><li> Ripetere il punto 2.2. </li><li> Incubare a 4 ° C per circa 15 ore. </li><li> Per il campione demetilasi, aggiungere 12,5 microlitri di 1 M Tris, pH 7,4 per placare la reazione riduttiva metilazione. </li></ol> 3. Spettrometria di massa MALDI <ol><li> Aggiungi Poros R2 20 perline micron alla reazione demetilasi, e raccogliere il ZipTips come descritto nei passaggi 1,3-1,6. </li></ol> Nota: Re-sospendere il controllo in 100 pl di tampone fosfato 50 mM, pH 7,4 e trattare come l&#39;altro campione partendo dal punto 3,1. <ol start="2"><li> Eluire il controllo e campioni di reazione demetilasi dalle ZipTips su una piastra di campioni utilizzando MALDI 2 microlitri di 33% saturared 2,5-diidrossibenzoico acido. Per preparare il 33% di acido 2,5-diidrossibenzoico, una soluzione satura di acido 2,5-diidrossibenzoico in 70% acetonitrile / 0,1% TFA e quindi diluire al 33% saturo in 70% acetonitrile / 0,1% TFA. Lasciare i campioni eluiti ad asciugare e cristallizzare. </li><li> Raccogliere spettri di massa con un MALDI-prOTOF spettrometro di massa PerkinElmerSciex o disponibili ad alta risoluzione spettrometro di massa MALDI 8,9,10. </li><li> Vedi spettri ed estrarre le aree dei picchi che utilizzano software PerkinElmerSciex TOFworks o software fornito dal produttore spettrometro di massa. </li><li> Verificare prodotti di reazione da MS 2 con un sistema di spettrometria di massa disponibile fornendo funzionalità di spettrometria di massa tandem. </li></ol> 4. Analisi dei dati <ol><li> Al fine di compensare la sovrapposizione isotopici creati utilizzando formaldeide pesante (Figura 1B), determinare l&#39;area di picco (A) rapporto di 13 C 2 e 13 C 4 isotopi relative per il picco monoisotopico per il campione di controllo, che non ha subito metilazione riduttiva (Fig. 2A). Eq 1: A 13C2 / A 12C = r 1 Eq 2: A 13C4 / A 12C = r 2 Questo vi darà il rapporto di peptide esistenti in questi stati isotopica (r 1 e r 2) specifico per il tuo esperimento e spettrometro di massa. </li><li> Utilizzare la r 1 r 2 e valori nelle seguenti formule per quantificare la quantità relativa di peptide esistente in ogni stato modificazione del campione reazione demetilasi (Fig. 2B): Eq 3: H3K4me2 = A 1 Eq 4: H3K4me = A 2 - r 1 (A 1) Eq 5: H3K4 = A 3 - r 2 (A 1) - [r 1 (A 2 - r 1 (A 1))] </li></ol> 5. Risultati rappresentativi Utilizzando LSD1, ci mostrano l&#39;efficacia di MassSQUIRM per quantificaredemetilazione lisina. Come descritto nella sezione Testo protocollo, abbiamo eseguito un saggio demetilasi con 125 ng di LSD1 e 0,25 mg di metil-istone H3 peptide (ARTKme2QTARKSTGGKAPRKQLYK-biotina). Il controllo e la reazione demetilasi campioni sono stati sottoposti a metilazione riduttiva e spettrometria di massa MALDI. La reazione di controllo che non ha subito metilazione riduttiva mostrato la busta isotopico tipico per questo peptide e fornito aree di picco di equazioni 1 e 2 (Figura 2A). Lo spettro di massa per la reazione demetilasi fornito aree di picco per 3-5 equazioni (Figura 2B). Utilizzando le aree dei picchi dal controllo e reazione demetilasi, abbiamo determinato che LSD1 demetilato il peptide per dare i seguenti prodotti di reazione: 33,7% di metil-Lys4, 42,3% mono-metil Lys4, e 24% non-metilata Lys 4. Fare riferimento a Blair et al. Per l&#39;analisi completa di LSD1 attività demetilasi nonché studi inibitori 6. Si noti che le variabili cambiano come tempo di reazione, enzimaticaconcentrazione e la concentrazione del substrato consentirà una approfondita analisi delle attività. <img alt="Figura 1" src="/files/ftp_upload/3604/3604fig1.jpg" /> Figura 1. Panoramica MassSQUIRM. (A) Un peptide N-terminale da istone H3 viene mostrato come un-(verde), mono-(rosso), o di-(blu) metilato a lisina 4. Variazione della composizione chimica di ciascun peptide porta a ionizzazione differenziale rendere complesso quantificazione. (B) metilazione riduttiva converte tutti i residui di lisina al di-metil stato che fa sì che tutti i peptidi per ionizzare in modo simile. L&#39;uso di formaldeide pesante nella reazione di metilazione riduttiva permette la conservazione dell&#39;identità della metilazione originale. Cerchi aperti indicano metilazione luce, mentre i cerchi chiusi indicano metilazione pesante. Modificato da Blair et al. 2011 6. <img alt="Figura 2" src="/files/ftp_upload/3604/3604fig2.jpg" /> Figure 2. MassSQUIRM può essere utilizzato per quantificare peptidi differenzialmente metilati. (A) di A-metilato sintetico istone H3 peptide (ARTKme2QTARKSTGGKAPRKQLYK-biotina) è stato analizzato mediante spettrometria di massa e rapporti di picco relativa al picco monoisotopico sono state rilevate come r 1 ed r 2 in equazioni 1 e 2. (B) Lo stesso peptide sintetico è stato incubato con 125 ng LSD1 in tampone demetilasi per due ore a 37 ° C. I campioni sono stati poi sottoposti ad analisi MassSQUIRM. Una popolazione mista di sovrapposizione picchi rappresentano tre diversi stati di metilazione come si vede nella figura 1B. Le aree sotto i picchi sono stati notati monoisotopico come A 1, A 2 e A 3. Aree dei picchi sono stati utilizzati nelle equazioni 3-5. Cerchi aperti indicano metilazione luce, mentre i cerchi chiusi indicano metilazione pesante. Modificato da Blair et al. 2011 6.

<ol>
	<li>Goldberg, A. D., Allis, C. D., Bernstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics:+a+landscape+takes+shape.">Epigenetics: a landscape takes shape.</a> Cell. 128, 635-638 (2007).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128, 693-705 (2007).</li><li>Blair, L. P., Cao, J., Zou, M. R., Sayegh, J., Yan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+Regulation+by+Lysine+Demethylase+5+(KDM5)+Enzymes+in+Cancer.">Epigenetic Regulation by Lysine Demethylase 5 (KDM5) Enzymes in Cancer.</a> Cancers (Basel). 3, 1383-1404 (2011).</li><li>Cole, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+for+histone-modifying+enzymes.">Chemical probes for histone-modifying enzymes.</a> Nat. Chem. Biol. 4, 590-597 (2008).</li><li>Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+demethylation+mediated+by+the+nuclear+amine+oxidase+homolog+LSD1.">Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.</a> Cell. 119, 941-953 (2004).</li><li>Blair, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MassSQUIRM:+An+assay+for+quantitative+measurement+of+lysine+demethylase+activity.">MassSQUIRM: An assay for quantitative measurement of lysine demethylase activity.</a> Epigenetics. 6, 490-499 (2011).</li><li>Rayment, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+myosin+subfragment-1:+a+molecular+motor.">Three-dimensional structure of myosin subfragment-1: a molecular motor.</a> Science. 261, 50-508 (1993).</li><li>Collom, S. L., Jamakhandi, A. P., Tackett, A. J., Radominska-Pandya, A., Miller, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CYP2E1+active+site+residues+in+substrate+recognition+sequence+5+identified+by+photoaffinity+labeling+and+homology+modeling.">CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling.</a> Arch. Biochem. Biophys. 459, 59-69 (2007).</li><li>Gradolatto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+Yta7+Regulates+Histone+Gene+Expression.">Saccharomyces cerevisiae Yta7 Regulates Histone Gene Expression.</a> Genetics. 179, 291-304 (2008).</li><li>Taverna, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yng1+PHD+finger+binding+to+histone+H3+trimethylated+at+lysine+4+promotes+NuA3+HAT+activity+at+Lysine+14+of+H3+and+transcripiton+at+a+subset+of+targeted+ORFs.">Yng1 PHD finger binding to histone H3 trimethylated at lysine 4 promotes NuA3 HAT activity at Lysine 14 of H3 and transcripiton at a subset of targeted ORFs.</a> Mol. Cell. 24, 785-796 (2006).</li></ol>

Non abbiamo nulla da rivelare.

MassSQUIRM è un metodo poco costoso e quantitative per l&#39;analisi complessiva dell&#39;attività di demethylases lisina coinvolti in mono-e di-metilazione. MassSQUIRM quantificazione offre non solo del prodotto della reazione, ma anche per gli intermedi. Questa analisi può essere utilizzato come un potente strumento per studiare il meccanismo di demethylases istone LSD1 e altri. Sarà anche utile per classificare molti enzimi recentemente scoperti demetilasi lisina come PHF8 e potrebbe essere utilizzato per alcuni ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> LSD1 </td><td> BPS Biosciences </td><td> 50100 </td></tr><tr><td> H3K4me2-biotina peptide </td><td> preparato in-house </td><td> nessuno </td></tr><tr><td> POROS R2 20 perline micron </td><td> Applied Biosystems </td><td> 1-1129-06 </td></tr><tr><td> C 18 ZipTip </td><td> Millipore </td><td> ZTC18M </td></tr><tr><td> Acido trifluoroacetico (TFA) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> acetonitrile </td><td> Pescatore </td><td> A996 </td></tr><tr><td> 2,5-diidrossibenzoico acido </td><td> Sigma </td><td> 85707 </td></tr><tr><td> Borano dimetilammina </td><td> Sigma </td><td> 180238 </td></tr><tr><td> isotopicamente pesante d 2-formaldeide </td><td> Cambridge Isotope Laboratories </td&gt; <td> DLM-805-20 </td></tr><tr><td> Tris </td><td> Pescatore </td><td> BP154 </td></tr><tr><td> KCl </td><td> Pescatore </td><td> BP366 </td></tr><tr><td> MgCl 2 </td><td> Pescatore </td><td> BP214 </td></tr><tr><td> Glicerina </td><td> Pescatore </td><td> G33 </td></tr><tr><td> Acido formico </td><td> Fluka </td><td> 06440 </td></tr><tr><td> Metanolo </td><td> Pescatore </td><td> A452 </td></tr><tr><td> Na-fosfato </td><td> Pescatore </td><td> BP329 </td></tr><tr><td> SpeedVac Concentrator </td><td> Dotto </td><td> DNA110 </td></tr><tr><td> MALDI-prOTOF Spettrometro di massa e TOFworks software </td><td> PerkinElmerSciex </td><td> nessuno </td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

Recentemente, i regolatori epigenetici sono stati scoperti come i giocatori chiave per molte diverse malattie 1-3. Come risultato, questi enzimi sono obiettivi primari di studi piccola molecola e sviluppo 4 farmaci. Molti regolatori epigenetici hanno solo recentemente è stato scoperto e sono ancora in fase di classificazione. Tra questi enzimi sono demethylases lisina che rimuovono gruppi metilici da lisine degli istoni ed altre proteine. A causa della natura di questa nuova classe di enzimi, pochi saggi sono stati sviluppati per studiare la loro attività. Questo è stato un blocco stradale sia per la classificazione e lo studio elevato throughput di demethylases istoni. Attualmente, saggi demetilasi esistono pochi. Quelli che esistono tendono ad essere di natura qualitativa e non possono contemporaneamente discernere tra i diversi stati di metilazione della lisina (Nazioni Unite-, mono-, di-e tri-). La spettrometria di massa è comunemente usato per determinare l&#39;attività demetilasi ma gli attuali dosaggi di massa spettrometria di farenon affrontare se peptidi differenzialmente metilati ionizzare in modo diverso. Ionizzazione dei peptidi metilati differenziale rende il confronto stati metilazione difficile e certamente non quantitativa (Figura 1A). Così test disponibili non sono ottimizzati per l&#39;analisi completa delle attività demetilasi. Qui si descrive un metodo chiamato MassSQUIRM (spettrometria di massa quantificazione utilizzando isotopica metilazione riduttiva) che si basa sulla metilazione riduttiva di gruppi amminici con formaldeide deuterato di costringere tutti lisine essere di-metilato, rendendoli così in sostanza la stessa specie chimiche e quindi ionizzare l&#39; stesso (figura 1B). La differenza chimica soltanto dopo la metilazione riduttiva è idrogeno e deuterio, che non influenza l&#39;efficienza di ionizzazione MALDI. Il dosaggio MassSQUIRM è specifico per i prodotti di reazione demetilasi con un-lisine, mono-o di-metilati. Il saggio è applicabile anche a methyltransferases lisina dando la same prodotti di reazione. Qui, si usa una combinazione chimica di metilazione riduttiva e spettrometria di massa MALDI per misurare l&#39;attività di LSD1, uno demetilasi lisina in grado di rimuovere di-e mono-metil gruppi, su un substrato peptidico sintetico 5. Questa analisi è semplice e facilmente suscettibili di qualsiasi laboratorio con accesso ad uno spettrometro di massa MALDI in laboratorio o attraverso un servizio di proteomica. Il saggio deve ~ 8 volte la gamma dinamica, ed è facilmente scalabile in formato piastra 5.

Watch this Scientific Journal Video about Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity at JoVE.com

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Presentiamo un metodo per utilizzare spettrometria di massa MALDI e chimica metilazione riduttiva per quantificare variazioni di metilazione lisina.

Applicazione di MassSQUIRM per le misure quantitative delle attività demetilasi lisina

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

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Biology

13.3K Views.  University of Arkansas for Medical Sciences. Presentiamo un metodo per utilizzare spettrometria di massa MALDI e chimica metilazione riduttiva per quantificare variazioni di metilazione lisina.

Video: Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

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 ...

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Biologia

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

Quantitativa, in tempo reale Analisi dell&#39;attività riparazione per escissione Base in lisati cellulari Utilizzando Molecular Beacons Lesione specifici

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Misure calcio citosolico nelle cellule epiteliali renali mediante citometria di flusso

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Analisi Specificità di proteine ​​lisina metiltransferasi Utilizzando SPOT Peptide Array

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Ricostituzione funzionale e attività Canale Misure di purificata di tipo selvatico e mutante CFTR proteine

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Sviluppo e caratterizzazione di<em&gt; In Vitro</em&gt; Microvasi Rete e misurazioni quantitative di endoteliale [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; E ossido nitrico Produzione

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

Applicazione di misura di elettrofisiologia di studiare l'attività dei trasportatori di Electro-neutro

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Quantificazione di site-specific Protein lisina acetilazione e stechiometria Succinylation usando la spettrometria totale di acquisizione dati-indipendente

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.