Biz kitle spektrometrik destek için UAMS Proteomik Tesis teşekkür ederim. Bu proje için finansman NIH hibe P20RR015569, P20RR016460 ve R01DA025755 tarafından sağlandı.

Bu protokol, Blair ve ark değiştirilir. 6. 1. LSD1 demetilasyonu Assay <ol><li> 20 ul&#39;lik bir son hacim içinde, demetilaz tampon içinde 0.25 g di-metil histon H3 peptit (ARTKme2QTARKSTGGKAPRKQLYK-biyotin) (50 mM Tris-Cl, pH 8.5, 50 mM KCI, 5 mM MgCl2,% 5 ile 125 ng rekombinant LSD1 kombine gliserol). Buna ek olarak, herhangi bir enzim ile bir peptid sadece kontrol gerçekleştirmek. Kontrol bölümü 3 ve indirgeyici metilasyon gerekmez. </li><li> 37 de 2 saat boyunca inkübe ° C. </li><li> Peptidler toplamak için, her bir örnek için 8 uL Póros R2, 20 mikron boncuklar ekleyin ve oda sıcaklığında 15 dakika boyunca ajite. Metanol 1 hacmine sahip boncuk 1 hacim süspanse ederek Poros boncuklar hazırlamak ve daha sonra% 5 formik asit /% 0.2 TFA içerisinde 10 hacim ekleyin. </li><li> Durumu iki ucu içine% 0.1 TFA 20 uL aspirasyon ve bir pipet ile geri dışarı iterek C 18 ZipTips. % 0,1 T ile iki kez yapınFA, dört adet 70% asetonitril ile kere /% 0.1 TFA, ve% 0.1 TFA ile yeniden dört kez. </li><li> ZipTips in C&#39;de 18 reçinesi arkasında boncuk bulamaç pipet ve bir pipet ile yoluyla hacmi iterek şartlandırılmış C&#39;de 18 ZipTips üzerine iki numune (kontrolü ve demetilaz reaksiyonu) yerleştirin. </li><li> % 0.1 TFA içerisinde 20 uL ile iki kez yıkanır ZipTips. </li><li> % 70 asetonitril /% 0.1 TFA içerisinde 40 uL içinde elüt edilmesi. </li><li> Bir SpeedVac konsantratör tamamen her seyreltici Lyophilize. </li></ol> 2. Redüktif Metilasyon Bu protokolün bir kısmı Blair ve ark modifiye edilir. Ve Rayment ve ark. 6,7. <ol><li> 50 mM fosfat tamponu, pH 7.4 100 ul içinde demetilaz Reaksiyon tekrar askıya. </li><li> Demetilaz reaksiyon, 15 mg / mL boran dimetilamin 8 uL ekleyin. Boran dimetilamin 15 mg / mL stok çözelti vermesi için, 50 mM fosfat tamponu, pH 7.4 içinde çözülür.Bu taze yapılmalıdır. </li><li> Demetilaz reaksiyon için, 250 mM döteryumlanmış formaldehit 16 uL ekleyin. 250 mM Döteryumlanmış formaldehid H 2 0 içinde stok seyreltilmesi ile hazırlanır. Bu taze yapılmalıdır. </li><li> 2 saat süreyle 4 ° C&#39;de inkübe edilir. </li><li> Adımları 2.2, 2.3 ve 2.4 tekrarlayın. </li><li> Adım 2.2 tekrarlayın. </li><li> 4 ° C&#39;de inkübe ~ 15 saat boyunca. </li><li> Demetilaz örnek için, 1M Tris, indirgeyici metilasyon reaksiyonu gidermek için pH 7.4 12.5 uL ekleyin. </li></ol> 3. MALDI Kütle Spektrometresi <ol><li> Demetilaz reaksiyon Poros R2 20 mikron boncuk ekleyin ve adımları 1,3-1,6 açıklandığı gibi ZipTips üzerinde toplamak. </li></ol> Not: 50 mM fosfat tamponu, pH 7.4 100 uL, kontrol yeniden askıya alma ve diğer örnek adım 3.1 başlayarak gibi davran. <ol start="2"><li> % 33 doyurmak 2 uL kullanılarak bir MALDI Örnek plakası üzerine ZipTips gelen kontrol ve demetilaz Reaksiyon numuneleri elüt edilmesid 2,5-dihidroksibenzoik asit. % 33 2,5-dihidroksibenzoik asit hazırlamak için,% 70 asetonitril /% 0.1 TFA içerisinde doymuş% 33 kadar seyreltik daha sonra% 70 asetonitril /% 0.1 TFA içerisinde 2,5-dihidroksibenzoik asit, doymuş bir çözelti yapmak ve. Yıkanan örnekleri kurulayın ve kristalize izin verin. </li><li> Bir PerkinElmerSciex MALDI-prOTOF kütle spektrometresi veya mevcut yüksek çözünürlüklü MALDI kütle spektrometresi 8,9,10 kullanarak kütle spektrumları toplayın. </li><li> Spektrumları görüntüleme ve kütle spektrometre üreticisi tarafından sağlanan PerkinElmerSciex TOFworks yazılım veya yazılım kullanarak pik alanları ayıklayın. </li><li> Tandem kütle spektrometresi yetenekleri sağlayan kullanılabilir bir kütle spektrometresi sistemi ile MS 2 ile reaksiyon ürünleri doğrulayın. </li></ol> 4. Veri Analizi <ol><li> Ağır formaldehid (Şekil 1B) kullanılarak hazırlandı izotopik örtüşme telafi etmek için, pik alanı 13 C&#39;de 2 ve 13 C&#39;de 4 izotopları relativ (A) oranının belirlenmesiindirgeyici metilasyon (Şekil 2A) uğramamıştır kontrol örneği için Monoizotopik pik e. Denklem 1: 13C2 / A 12C = r 1 Denklem 2: A 13C4 / A 12C = r 2 Bu sizin deney ve kütle spektrometresi özgü bu izotop devletlerin mevcut peptid oranı (r 1 ve r 2) verecektir. </li><li> Demetilaz reaksiyonu Örnek olarak, her durumda modifikasyon (Şek. 2B) mevcut peptid görece miktarı ölçülmesi için aşağıdaki formüllerde R 1 ve R2 değerlerinin kullanımı: Eşitlik 3: H3K4me2 = A 1 Eq 4: H3K4me = A 2 - r 1 (A 1) Denklem 5: H3K4 = A 3 - r 2 (A 1) - [r 1 (A 2 - r 1 (A 1))] </li></ol> 5.. Temsilcisi Sonuçlar LSD1 kullanarak, kantifikasyonunda MassSQUIRM etkinliğini göstermeklisin demetilasyon. Gibi Protokolü Text bölümünde açıklandığı üzere, bu LSD1 125 ng ve 0.25 g di-metil histon H3 peptit (ARTKme2QTARKSTGGKAPRKQLYK-biotin) ile bir demetilaz deneyi gerçekleştirilir. Kontrolü ve demetilaz Reaksiyon numuneleri indirgeyici metilasyonu ve MALDI kütle spektrometrisi tabi tutuldu. Indirgeyici metilasyon uğramadı kontrol reaksiyonu Bu peptid için tipik izotopik zarfı gösterdi ve denklemleri 1 ve 2 (Şekil 2A) için pik alanları sağlamıştır. Demetilaz reaksiyon için kütle spektrumu denklemler 3-5 (Şekil 2B) için pik alanı temin. Kontrolü ve demetilaz reaksiyondan pik alanları kullanarak, LSD1 aşağıdaki reaksiyon ürünleri vermek üzere peptid demetillenmiş belirlenmiştir:% 33.7 di-metil Lys4,% 42.3 mono-metil Lys4, ve% 24 un-metillenmiş Lys 4. Blair ve ark bakınız. Tam LSD1 demetilaz aktivitesinin analizi gibi inhibitörü çalışmaları için 6. Not böyle reaksiyon süresi, enzim olarak değişen değişkenlerkonsantrasyonu ve substrat konsantrasyonu faaliyet derinlemesine bir analiz için izin verecektir. <img alt="Şekil 1" src="/files/ftp_upload/3604/3604fig1.jpg" /> Şekil 1. MassSQUIRM bakış. (A) histon H3 bir peptidin N-terminali olarak gösterilir un-(yeşil), mono-(kırmızı), ya da di-(mavi), lisin 4 azından metillenmiş. Her peptid kimyasal bileşiminin değişimiyle miktar karmaşık hale diferansiyel iyonizasyon yol açar. (B) indirgeyici metilasyon tüm peptidler benzer şekilde iyonize neden olur di-metil durumuna tüm lisin artıkları dönüştürür. Indirgeyici metilasyon reaksiyonu ağır formaldehit kullanımı metilasyon orijinal kimlik retansiyon sağlar. Kapalı daireler ağır metilasyon işaret ederken açık daireler ışık metilasyon gösterir. Blair ve ark. 2011 6. modifiye edilmiştir. <img alt="Şekil 2" src="/files/ftp_upload/3604/3604fig2.jpg" /> Figüre 2. MassSQUIRM diferansiyel olarak metillenmiş peptidler ölçmek için kullanılabilir. (A) bir di-metillenmiş sentetik histon H3 peptit (ARTKme2QTARKSTGGKAPRKQLYK-biyotin) R 1 ve denklemler 1 ve 2 &#39;de R 2 olarak kaydedildi kütle spektrometrisi ve Monoizotopik pik göre pik oranları kullanılarak analiz edilmiştir. (B) aynı sentetik peptid, 37 ° C de iki saat süreyle demetilaz tampon içinde 125 ng LSD1 ile inkübe edildi Örnekler daha sonra, analizi MassSQUIRM tabi tutuldu. Zirveleri örtüşen karışık bir nüfus olarak Şekil 1B görülen üç farklı metilasyon devletleri temsil eder. Monoizotopik Pik Alanları A 1, A 2 ve A 3 olarak kaydedildi. Pik alanları denklemleri 3-5 kullanılmıştır. Kapalı daireler ağır metilasyon işaret ederken açık daireler ışık metilasyon gösterir. Blair ve ark Modifiye. 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>

Biz ifşa hiçbir şey yok.

MassSQUIRM mono-ve di-metilasyon dahil lisin demethylases aktivitesinin analizi için kapsamlı bir ucuz ve niceliksel bir yöntemdir. MassSQUIRM reaksiyon ürününün aynı zamanda ara ürün için sadece kantitatif sunmaktadır. Bu testte LSD1 ve diğer histon demethylases mekanizması okuyan güçlü bir araç olarak kullanılabilir. Aynı zamanda böyle PHF8 olarak yeni keşfedilen lisin demetilaz enzimler sınıflandırmak ve belirli metil enzimleri için kullanılabilecek için yararlı olacaktır.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> LSD1 </td><td> BPS Biosciences </td><td> 50100 </td></tr><tr><td> H3K4me2-biotin peptit </td><td> in-house hazırlanır </td><td> hiçbiri </td></tr><tr><td> Poros R2 20 mikron boncuk </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> Trifloroasetik asit (TFA) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> asetonitril </td><td> Balıkçı </td><td> A996 </td></tr><tr><td> 2,5-dihidroksibenzoik asit </td><td> Sigma </td><td> 85707 </td></tr><tr><td> Boran dimetilamin </td><td> Sigma </td><td> 180238 </td></tr><tr><td> izotopça ağır d 2-formaldehit </td><td> Cambridge İzotop Laboratuvarı </td&gt; <td> DLM-805-20 </td></tr><tr><td> Tris </td><td> Balıkçı </td><td> BP154 </td></tr><tr><td> KCl </td><td> Balıkçı </td><td> BP366 </td></tr><tr><td> MgCl2 </td><td> Balıkçı </td><td> BP214 </td></tr><tr><td> Gliserin </td><td> Balıkçı </td><td> G33 </td></tr><tr><td> Formik asit </td><td> Fluka </td><td> 06440 </td></tr><tr><td> Metanol </td><td> Balıkçı </td><td> A452 </td></tr><tr><td> Na-fosfat </td><td> Balıkçı </td><td> BP329 </td></tr><tr><td> SpeedVac Konsantratörü </td><td> Bilgin </td><td> DNA110 </td></tr><tr><td> MALDI-prOTOF kütle spektrometresi ve TOFworks yazılım </td><td> PerkinElmerSciex </td><td> hiçbiri </td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

Son zamanlarda, epigenetik düzenleyiciler 1-3 çok farklı hastalıklara kilit oyuncular olarak tespit edilmiştir. Sonuç olarak, bu enzim küçük molekül çalışmalar ve ilacın geliştirilmesi 4 ana hedefi vardır. Birçok epigenetik regülatörler son zamanlarda saptanmış olması ve sınıflandırılmaktadır süreci halen devam etmektedir. Bu enzimler arasında histonlar ve diğer proteinler üzerindeki lysines gelen metil grupları kaldırmak lisin demethylases vardır. Enzimlerin bu sınıfın yeni niteliği nedeniyle, birkaç testler faaliyetlerini incelemek için geliştirilmiştir. Bu sınıflandırma ve histon demethylases yüksek verimlilik çalışması hem de yol bloğu olmuştur. Günümüzde çok az demetilaz testler var. Var yaptığımız bu doğada nitel olma eğilimindedir ve aynı anda farklı lizin metilasyon devletler arasında ayırt edemez (un-, mono-, di-ve tri-). Kütle spektrometresi yaygın demetilaz etkinliğini belirlemek için kullanılan ancak mevcut kitle spektrometrik deneyleri yapmak edilirayirt metillenmiş peptidler farklı iyonize olmadığına ilişkin. Metillenmiş peptidlerin Diferansiyel iyonlaşma metilasyon devletler zor ve (Şekil 1A) kesinlikle niceliksel değil karşılaştırma yapar. Böylece mevcut testlerin demetilaz faaliyet kapsamlı bir analiz için optimize edilmiş değildir. Burada, böylece esas olarak aynı kimyasal maddeleri hale getirerek, tüm lysines di-metillenmiş olmak zorlamak için döteryumlanmış formaldehit ile amin gruplarının indirgeyici metilasyon dayanmaktadır MassSQUIRM olarak adlandırılan bir yöntem (izotopik indirgeyici metilasyon kullanılarak kütle spektrometrik kantitatif) ve dolayısıyla tarif iyonize aynı (Şekil 1B). Indirgeyici metilasyon izleyerek sadece kimyasal fark hidrojen ve MALDI iyonizasyon verimliliği etkilemez döteryum vardır. MassSQUIRM assay un-, mono-veya di-metillenmiş lysines ile demetilaz reaksiyon ürünleri için kesin. Assay da sa veren lisin methyltransferases için de geçerlidirBana reaksiyon ürünleri. Burada, indirgeyici bir metilasyon kimya ve MALDI kütle spektrometrisi LSD1 aktivitesini ölçmek için, bir sentetik peptid substrat üzerindeki 5, di-ve mono-metil grupları çıkarma yeteneğine sahip bir lisin demetilaz. Bir kombinasyonunu kullanmak Bu test kolay ve laboratuar veya bir proteomik tesisi yoluyla bir MALDI kütle spektrometresi erişimi olan herhangi bir laboratuvar kolayca tâbidir. Assay ~ 8 kat dinamik aralığı vardır ve plaka formatında 5 için kolayca ölçeklenebilir.

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

Biz lisin metilasyon değişiklikleri ölçmek için MALDI kütle spektrometresi ve indirgeyici metilasyon kimya kullanmak için bir yöntem sunuyoruz.

Lizin demetilaz Faaliyet Kantitatif Ölçümler MassSQUIRM Uygulaması

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 Görüntüleme.  University of Arkansas for Medical Sciences. Biz lisin metilasyon değişiklikleri ölçmek için MALDI kütle spektrometresi ve indirgeyici metilasyon kimya kullanmak için bir yöntem sunuyoruz.

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.

Etkinlik Ölçüm Bir Testi Escherichia coli Lizin Decarboxyase Đndüklenebilir

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 ...

Biyoloji

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.

Lezyon özgü Moleküler Beacon kullanılması Hücre lizatları ve Base Eksizyon Onarımı Faaliyet kantitatif, gerçek zamanlı Analizi

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.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<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.

Sitometrisi ile Böbrek Hücrelerine sitozolik kalsiyum Ölçümleri

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.

SPOT Peptit Diziler Protein Lizin Methyltransferases Özgünlüğü Analizi

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.

Saf Yabanitip ve Mutant CFTR Protein Fonksiyonel Sulandırma ve Kanal Aktivite Ölçümleri

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.

Gelişim ve karakterizasyonu<em&gt; İn Vitro</em&gt; Mikrodamar Ağ ve Endotel kantitatif ölçümleri [Ca<sup&gt; 2+</sup&gt;]<sub&gt; I</sub&gt; Ve Nitrik Oksit Üretimi

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.

Elektrofizyoloji ölçüm uygulanması elektro-tarafsız taşıyıcılar etkinlik çalışması için

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.

Siteye özel Protein lizin asetilasyon ve Succinylation veri bağımsız edinme kütle spektrometresi kullanılarak Stoichiometry miktar

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.