Biz istiyoruz AV Lefkofsky Aile Vakfı / Liz ve Eric Lefkofsky Yenilik Araştırma Ödülü - Burada anlatılan proje NIH / NCI (NCI-R01CA182506-01A1) bir hibe ile yanı sıra Robert H. Lurie Kapsamlı Kanser Merkezi tarafından desteklenen eleştirel okuma ve bu taslağın düzenlenmesi için laboratuvara üyeleri (Carol O&#39;Callaghan ve Elizabeth Anne Wayne) teşekkür etmek.

İn Vitro deasetilasyonu Tahlili 1. <ol><li> SIRT2 saflaştırılması <ol><li> % 10 FBS ve antibiyotikler olan 8 ml DMEM içinde kültürlenir ve yaklaşık% 70 konfluent ertesi gün için 37 ° C,% 5 CO2 doku kültürü inkübatöründe yetiştirilen HEK 293T hücreleri 10 cm&#39;lik kültür çanağı hazırlayın. </li><li> Ertesi gün, ko-transfeksiyonu 4 ug pCDH-puro-GFP SIRT2-Bayrak (laboratuarımızda yapılan Lentivector), 4 ug pCMV- dR8.2 dvpr (ambalaj vektör) ve 0.5 ug pCMV-VSV-G (zarf vektör) 3 ul PEİ / ug DNA arasında bir oranda 18 kullanılarak polietilenimin (PEI). </li><li> 12 saat sonra ortam değiştirin. </li><li> 36-48 saat sonra süpernatant toplayın. </li><li> 4 ° C&#39;de 5 dakika boyunca 1000 x g&#39;de santrifüj ile çöpleri çıkarın. </li><li> 0.22 um filtre aracılığıyla süzüldükten sonra SIRT2 lentivirüs 1 ml&#39;lik eş payları yapabilir ve -80 ° C&#39;de saklayın. </li><li> Virüs kullanılmadığında buz üzerinde çözülme SIRT2 lentivirüs (-80 ° C&#39;de virüs depolamak önemlidir). </li><li> 8 ug / ml polibren mevcudiyetinde lentivirüs 1 ml% 80-90 ortak akışlı HEK 293T hücreleri, 6 gözlü bir plaka Infect. </li><li> Bir sonraki gün (24 saat kadar), 37 ° C kuluçka makinesi içinde, virüs ile enfekte olmuş hücreler yerleştirin. </li><li> 24 saat sonra orta değiştirin. </li><li> enfeksiyondan 48 saat sonra, bir floresan mikroskop altında GFP pozitif hücrelerini tespit ederek enfeksiyon verimliliğini kontrol edin. </li><li> Enfeksiyon etkinliği iyi ise (en az% 40-50 enfekte olmuş hücreler), kültür ortamı hücreleri ifade eden stabil SIRT2 seçmek için 2 ug / ml puromisin içeren orta yerine. Not: Bu yaklaşık 2 hafta sürer ve orta her 3-4 günde bir değiştirilir. Enfekte olmayan hücreler SIRT2 lentivirüs büyümesi ile seçim süresi ve sadece enfekte olmuş hücreler üzerinde ölmektedir. </li><li> Purif başlamadan önce,% 10 SDS-PAGE jeli üzerinde toplam proteinin 40 ug çalıştırdıktan sonra (1000 sulandırma 1) bir anti-Flag antikor kullanılarak western blotting ile Flag-işaretli SIRT2 ekspresyonunu analizication işlemi (önerilir) 19,20. </li><li> 1. aktif SIRT2, bölünmüş hücrelerinin saflaştırılması için: kararlı bir şekilde aşırı eksprese eden Flag-SIRT2 HEK 293T hücrelerinin yeterli yemekler için, tripsinizasyon ile 3 oranında (on 10 cm&#39;lik kaplar, daha sonraki deneyler için yeterli proteinin saflaştırılmasını sağlar). <ol><li> , Hücreleri tripsinize kültür ortamı çıkarın ve steril DPBS ile hücrelerin durulanarak artık serum ortadan kaldırmak için. Yavaşça, hücre tekli tabakası kapsayan 30-45 saniye boyunca oda sıcaklığında inkübe için% 0.25 tripsin-EDTA çözeltisi 2.5 ml. hücreleri ayırmak için başlayana kadar Tripsin-EDTA çözeltisi uzaklaştırıldıktan sonra, 37 ° C&#39;de inkübe edin. Sonra kültür ortamı eklemek ve yeni yemekler hücreleri aktarmak. </li></ol></li><li> 4 ° C&#39;de 10 dakika boyunca 1000 x g&#39;de santrifüjleme ile, ardından tripsinizasyon ile hücreler PBS içinde iki kez hücreleri yıkama ve toplama, kültür ortamı çıkarın. </li><li> 500 ul liziz tamponu A (20 mM HEPES pH 7.9, 18 mM KCI, 0.2 mM EGTA, 1.5 mM MgCl2 içinde Hücre topağınıBir proteaz önleyicileri kokteyli ve sürekli dönme altında 4 ° C&#39;de 30 dakika boyunca 1 uM TSA içeren% 20 gliserol,% 0.1 NP-40). </li><li> yeni bir tüpe 15, 4 ° C&#39;de dakikada transfer süpernatan 14,000 x g&#39;de santrifüjleyin. </li><li> Aşağıda tarif edildiği gibi boncuklar agaroz konjuge edilmiş bir anti-Flag antikor kullanılarak imüno gerçekleştirin. <ol><li> Protein lizat (10-20 mg toplam protein) için boncuk agaroz konjüge anti-Flag antikor 200-300 ul ekle. </li><li> sabit çalkalama ile 4 ° C&#39;de gece boyunca inkübe edilir. </li><li> 4 ° C&#39;de 5 dakika boyunca 1000 x g&#39;de santrifüj ile imüno-toplayın. </li><li> 4 ° C&#39;de 5 dakika boyunca 1000 x g&#39;de pelet 10 mi liziz tamponu A ile 5 kez yıkayın. Her yıkamadan sonra, boncuklar pelet ve tampon A ile süpernatant yerine </li></ol></li><li> (Proteaz inhibitörleri kokteyl ve 1 PBS uM TSA dahil) 1x Bayrak peptid çözeltisi hazırlayın. </li><li> 1x Bayrak peptidi 500 ul ekleyinagaroz tanelerine e çözeltisi ve sabit bir dönme altında 4 ° C&#39;de 30 dakika inkübe edilir. </li><li> 4 ° C&#39;de 2 dakika boyunca 6000 x g&#39;de santrifüj ile yüzer toplayın. </li><li> önceki iki adımı tekrarlayın. </li><li> 4 ° C&#39;de 1 dakika boyunca 15,000 x g&#39;de filtre tüpleri ve santrifüjleme ile süpernatandan boncuk çıkarın. </li><li> Nihai protein konsantrasyonu 1 ug kadar / bir ultrafiltrasyon membranı kullanılarak ul elüt örneğini konsantre edilir. </li><li> Ekstrakte edilen örnek 1 ug kullanılarak gerçekleştirilmiştir elektroforez ile saflaştırma işlemi kontrol edin. </li><li> Bir ticari olarak temin edilebilen boyama solüsyonu ile Leke jeli SIRT2 arıtma teyit etmek için. </li><li> -80 ° C&#39;de saflaştırılmış SIRT2 tutun. </li></ol></li><li> Asetile protein alt-tabaka saflaştırılması <ol><li> yaklaşık% 70 konfluent ertesi gün için HEK 293T hücreleri ile 10 cm x 10 cm kültür tabaklarında hazırlayın. </li><li> Bir Bayrağın 5 mikrogram ile ertesi gün, ko-transfeksiyonu hücreleri plazmid vektörü İfade Eden etiketliG-protein alt-tabaka ve 3 ul PEİ / ug DNA arasında bir oranda PEI kullanarak proteini asetillenmesi belirli asetil-transferaz 5 ug. Not: Belirli bir asetil transferaz bilinmiyorsa, örneğin P300, CBP, GCN5, Tip60 ve PCAF şekilde bilinmektedir histon asetil-transferaz (şapkalar) oluşan bir karışım ile birlikte transfekte edilmesi. </li><li> 12 saat sonra orta değiştirin. </li><li> gün hücrelerini parçalayan önce, deasetilazları inhibe ederek protein substrat asetilasyon seviyelerini en üst düzeye çıkarmak için TSA / NAM içeren ortamı ile kültür ortamı değiştirerek gecede 1 uM TSA ve 2 mM nikotinamid (NAM) ile hücre tedavisi. </li><li> Transfeksiyondan 48 saat sonra, kültür ortamı çıkarın, 4 ° C&#39;de 10 dakika boyunca 1000 x g&#39;de santrifüjleme ile, ardından tripsinizasyon ile hücreler PBS içinde iki kez hücreleri yıkama ve toplar. </li><li> çanak başına 500 ul liziz tamponu A Hücre topağını, (20 mM HEPES pH 7.9, 18 mM KCI, 0.2 mM EGTA, 1.5 mM MgCl2,% 20 gliserol,% 0.1 NP-40), bir proteaz I dahil olmak üzerenhibitors kokteyli, 1 uM TSA ve sürekli dönme altında 4 ° C&#39;de 30 dakika süre ile 2 mM Cum. </li><li> yeni bir tüpe 15, 4 ° C&#39;de dakikada transfer süpernatan 14,000 x g&#39;de santrifüjleyin. </li><li> Aşağıda tarif edildiği gibi boncuklar agaroz konjuge edilmiş bir anti-Flag antikor kullanılarak imüno gerçekleştirin. <ol><li> Protein lizat (10-20 mg toplam protein) için boncuk agaroz konjüge anti-Flag antikor 200-300 ul ekle. </li><li> sabit çalkalama ile 4 ° C&#39;de gece boyunca inkübe edilir. </li><li> 4 ° C&#39;de 5 dakika boyunca 1000 x g&#39;de santrifüj ile imüno-toplayın. </li><li> 4 ° C&#39;de 5 dakika boyunca 1000 x g&#39;de pelet 10 mi liziz tamponu A ile 2 kez yıkanır. Her yıkamadan sonra, boncuklar pelet lisiz tampon A ile süpernatant yerine </li><li> 4 ° C&#39;de 5 dakika boyunca 1000 x g&#39;de NAM olmayan pelet 10 mi liziz tamponu A ile 3 kez yıkanır. Her yıkamadan sonra, boncuk pelet ve bu importa olan lizis tamponu A ile süpernatant yerinent deasetilasyon reaksiyona herhangi NAM üzerine taşımak için değil. </li></ol></li><li> (Proteaz inhibitörleri ve PBS içinde 1 uM TSA dahil) 1x Bayrak peptid çözeltisi hazırlayın. </li><li> agaroz tanelerine 1x Bayrak peptid çözeltisinin 500 ul ilave edin ve sabit dönme altında 4 ° C&#39;de 30 dakika inkübe edilir. </li><li> 4 ° C&#39;de 2 dakika boyunca 6000 x g&#39;de santrifüj ile yüzer toplayın. </li><li> önceki iki adımı tekrarlayın. </li><li> 4 ° C&#39;de 1 dakika boyunca 15,000 x g&#39;de filtre tüpleri ve santrifüjleme ile süpernatandan boncuk çıkarın. </li><li> Protein konsantrasyonu 1 mg / ul kadar bir ultrafiltrasyon membranı kullanılarak, yıkanmış numune konsantre edilir. </li><li> % 4-12 SDS-PAGE jeli üzerinde elüt örnek 1 ul çalıştırarak elektroforez gerçekleştirin. </li><li> (: 1000 sulandırma 1) ve protein-alt-tabaka (dilüsyon kullanılan spesifik antikor bağlıdır) Western Ac-K karşı antikorlar kullanılarak blotting ile protein alt-tabakanın asetilasyonu teyit edin. </li><li> puri tutunğı -80 ° C&#39;de protein alt-tabakanın asetile. </li></ol></li><li> İn Vitro deasetilasyonu Reaksiyon <ol><li> Deasetilasyon tamponu B hazırlama (50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 1 mM MgCI2, 0.5 uM TSA) </li><li> 20 | il son hacme (Tablo 1) farklı tüplerinde 3 farklı tepkilere hazırlayın. </li><li> (Isteğe bağlı) SIRT2 protein-substrat entasetilleştirmeyi onaylamak için ek reaksiyonlar ekleyin. (NAM yerine deasetilaz aktif SIRT2 reaksiyonu (a pCDH-Puro-GFP SIRT2 H187Y -Flag Lentivector kullanılarak sonra 1.1&#39;de tanımlanan protokol kullanılarak yapılabilir) saflaştırılmış deasetilasyonu boş mutant SIRT2 2 ug ekleme ya da 10 mM nikotinamid ekleme ) SIRT2 deasetilasyonu aktivitesini inhibe etmek üzere reaksiyona (Tablo 1). </li><li> sabit bir ajitasyon altında 30 ° C de 3 saat süre ile reaksiyonlar inkübe edin. </li><li> f 2x numune tampon maddesi 20 ul ilave ederek reaksiyonu durdurun ve örnekleri kaynatınveya 95 ° C&#39;de 10 dakika. </li><li> % 4-12 SDS-PAGE jeli üzerinde reaksiyon karışımının çalıştırarak elektroforez gerçekleştirmek ve bir anti Ac-K antikoru (1: 1000 seyreltme) kullanan western blotting ile protein alt-tabaka ile asetilleme durumu tespit 19,20. </li></ol></li></ol> İn Vivo deasetilasyonu Tahlili 2. <ol><li> Kültürlenmiş Hücrelerde SIRT2 aşırı ekspresyonu <ol><li> HEK 293T hücreleri ile 10 cm kültür tabaklarında stabil olarak pCDH-Puro-GFP boş vektör aşırı ifade hazırlayın pCDH-Puro-GFP SIRT2-Flag ve pCDH-Puro-GFP SIRT2 H187Y -Flag (1.1&#39;de tarif edildiği gibi) yaklaşık 70 olmak % konfluent. </li><li> Seçenek olarak ise, hücreler, yaklaşık% 70 konfluent 10 cm kültür tabaklarında hazırlama ve 3 ul PEİ / ug DNA arasında bir oranda PEI ile Yukarıda zikredilen vektörlerin 5 ug kullanarak geçici aşırı ekspresyonunu gerçekleştirmek. </li><li> Flag-t aşırı ekspresyonu göstermek için: (1000 seyreltme 1) bir anti-Flag antikor kullanılarak toplam hücresel ekstrelerinin Western blotting işleminin gerçekleştirilmesiSIRT2 19 agged. </li></ol></li><li> Kültür Hücrelerde demonte SIRT2 RNA Girişim <ol><li> yaklaşık% 70 konfluent ertesi gün için HEK 293T hücreleri 10 cm&#39;lik kültür çanağı hazırlayın. </li><li> Sonraki gün, birlikte transfekte 4 ug pLKO1-Puro-SH SIRT2 (Lentivector) 19, 4 ug pCMV- dR8.2 dvpr (ambalaj vektör) ve 0.5 ug pCMV-VSV-G (kaplama vektör) 18 kullanılarak polietilenimin (PEI) 3 ul PEİ / ug DNA arasında bir oranda. Not: SIRT2 aşağı vurma için, RNAi Konsorsiyumu (TRC) tarafından tasarlanan pLKO.1 lentiviral vektör basit saç tokası shRNAs kullanın. </li><li> ShSIRT2 lentivirüs üretmek SIRT2 demonte hedef hücreleri enfekte etmek için 1.1 &#39;de tarif edilen prosedür takip edin. </li><li> % 10 SDS-PAGE jeli üzerinde toplam proteinin 40 ug çalıştırdıktan sonra etkin SIRT2 demonte göstermek için: (1000 sulandırma 1) bir anti-SIRT2 antikoru kullanılarak toplam hücresel ekstrelerinin Western blotting işleminin gerçekleştirilmesi. </li><li> Seçenek olarak ise,Hücreler yaklaşık% 70 konfluent ile 10 cm kültür yemekleri hazırlamak ve üreticinin talimatlarına siRNA&#39;lara kullanarak SIRT2 geçici demonte yaparlar. </li><li> % 10 SDS-PAGE jeli üzerinde toplam proteinin 40 ug çalıştırdıktan sonra etkin SIRT2 demonte göstermek için: (1000 sulandırma 1) bir anti-SIRT2 antikoru kullanılarak toplam hücresel ekstrelerinin Western blotting işleminin gerçekleştirilmesi. </li></ol></li><li> Immunoprecipitation ve Bağışıklık Kültürlü Hücrelerde Asetillendirilmiş Seviyeleri Detect <ol><li> 12 saat SIRT2 fazla sentezleyen ya da hücrelerden hücre pelletleri elde edilmesinden önce olmayan Sınıf III histon deasetilazların inhibe etmek için kültür ortamına 1 uM TSA ekleyin (yukarıdaki 2.1) ya da SIRT2 hücreleri (yukarıda 2.2) nakavt. </li><li> 4 ° C&#39;de 10 dakika boyunca 1000 x g&#39;de santrifüjleme ile, ardından tripsinizasyon ile hücreler PBS içinde iki kez hücreleri yıkama ve toplama, kültür ortamı çıkarın. </li><li> 10 mi liziz tamponu A (20 mM HEPES pH 7.9, 18 mM KCI, 0.2 mM EGTA, 1.5 eklememM MgCl2,% 20 gliserol, bir proteaz inhibitörleri kokteyli, 1 uM TSA ve 2 mM NAM içeren% 0.1 NP-40). </li><li> Sabit dönme altında, 30 dakika boyunca 4 ° C&#39;de inkübe edin. </li><li> 4 ° C&#39;de 15 dakika boyunca 20,000 x g&#39;de santrifüj ile süpernatantlar toplayın. </li><li> Bir Bradford protein tahlili kullanılarak protein konsantrasyonu hesaplanır. </li><li> Bir referans olarak liziz tamponu A kullanarak Tablo 2 kullanılarak 1 ml lik bir nihai hacim içinde Aktarım yeni tüplere toplam proteinin, 1 mg ya da aşırı ifade SIRT2 ya SIRT2 demonte sonra hücrelerden protein örnekleri hazırlanır. </li><li> Protein A / G ile birlikte belirli bir protein alt-tabakaya karşı bir antikor kullanılarak bir imüno-gerçekleştirme (40 ul boncuk çamurunun önerilir). Seçenek olarak ise, bütün asetile proteinleri immünopresipitasyon boncuk agaroz konjuge edilmiş bir anti Ac-K antikoru (20 ul boncuk çamuru genellikle yeterlidir) kullanın. </li><li> Consta altında 4 ° C&#39;de inkübe edinnt rotasyon gecede. </li><li> 4 ° C&#39;de 2 dakika boyunca 1000 x g&#39;de santrifüj ile imüno-toplayın. </li><li> 4 ° C&#39;de 2 dakika boyunca 1000 x g&#39;de boncuk 1 ml tampon A ile 5 kez yıkayın. </li><li> 2x numune tampon 40 ul yeniden süspanse boncuk. </li><li> 95 ° C&#39;de 10 dakika boyunca kaynatılır örnekleri. </li><li> elüt örnekleri aktarırken boncuk bozmadan% 4-12 SDS-PAGE jeli üzerinde elüt örnekleri (aşama 2.3.12 40 ul) çalıştırarak elektroforez gerçekleştirin. </li><li> bir protein alt-tabaka-özel antikor immüno-çökeltme için kullanıldı halinde (1.000 sulandırma 1), ya da protein alt-tabakaya karşı spesifik bir antikor (tavsiyeler izleyin, bir anti-Ac-K antikoru kullanılarak Western blotting ile protein alt-tabaka asetillenmiş düzeylerini algılar Anti Ac-K boncuklar immüno-çökeltme için kullanıldı, spesifik bir antikor ile ilgili seyreltme). </li></ol></li></ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+histone+deacetylases+in+development+and+physiology:+implications+for+disease+and+therapy.">The many roles of histone deacetylases in development and physiology: implications for disease and therapy.</a> Nature Reviews. Genetics. 10, 32-42 (2009).</li><li>Finkel, T., Deng, C. X., Mostoslavsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+biology+and+physiology+of+sirtuins.">Recent progress in the biology and physiology of sirtuins.</a> Nature. 460, 587-591 (2009).</li><li>Houtkooper, R. H., Pirinen, E., Auwerx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuins+as+regulators+of+metabolism+and+healthspan.">Sirtuins as regulators of metabolism and healthspan.</a> Nature reviews. Molecular Cell Biology. 13, 225-238 (2012).</li><li>Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+landscape+of+lysine+acetylation+links+metabolism+and+cell+signalling.">The growing landscape of lysine acetylation links metabolism and cell signalling.</a> Nature Reviews. Molecular Cell Biology. 15, 536-550 (2014).</li><li>Stewart, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-delivered+stable+gene+silencing+by+RNAi+in+primary+cells.">Lentivirus-delivered stable gene silencing by RNAi in primary cells.</a> RNA. 9, 493-501 (2003).</li><li>Kim, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRT2+maintains+genome+integrity+and+suppresses+tumorigenesis+through+regulating+APC/C+activity.">SIRT2 maintains genome integrity and suppresses tumorigenesis through regulating APC/C activity.</a> Cancer Cell. 20, 487-499 (2011).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRT2+directs+the+replication+stress+response+through+CDK9+deacetylation.">SIRT2 directs the replication stress response through CDK9 deacetylation.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 13546-13551 (2013).</li><li>Weinert, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+mapping+of+the+Drosophila+acetylome+demonstrates+a+high+degree+of+conservation+of+lysine+acetylation.">Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation.</a> Science Signaling. 4, ra48 (2011).</li><li>Still, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+mitochondrial+acetylation+dynamics+highlights+prominent+sites+of+metabolic+regulation.">Quantification of mitochondrial acetylation dynamics highlights prominent sites of metabolic regulation.</a> The Journal of Biological Chemistry. 288, 26209-26219 (2013).</li><li>Sadoul, K., Wang, J., Diagouraga, B., Khochbin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tale+of+protein+lysine+acetylation+in+the+cytoplasm.">The tale of protein lysine acetylation in the cytoplasm.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 970382 (2011).</li><li>Preble, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+and+purification+of+mitochondria+for+transplantation+by+tissue+dissociation+and+differential+filtration.">Rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration.</a> Journal of Visualized Experiments : JoVE. , e51682 (2014).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> Journal of Visualized Experiments : JoVE. , (2011).</li><li>He, W., Newman, J. C., Wang, M. Z., Ho, L., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+sirtuins:+regulators+of+protein+acylation+and+metabolism.">Mitochondrial sirtuins: regulators of protein acylation and metabolism.</a> Trends in Endocrinology and Metabolism: TEM. 23, 467-476 (2012).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Recent high throughput proteomic studies have established acetylation as a widespread PTM found not only in nucleus but also in cytoplasm and mitochondria 7,8,21-23. Taking into account the likelihood that many more acetylated proteins and sites might have been not detected due to several reasons, such as specificity of the anti-Ac-K antibodies used, the low abundance of the acetylated proteins, and the transient nature of the PTM, it is safe to predict that more acetylated proteins remain to be discovered in ...

Post-translational modifications (PTMs) regulate cell signaling networks allowing cells to rapidly respond to internal and external signals. Over the last few decades, many different PTMs playing a pivotal role in diverse processes have been identified but only a few have been studied extensively, such as phosphorylation, acetylation and ubiquitination 1-3. Focusing on acetylation, Allfrey et al. were the first to propose a role for histone acetylation in regulating gene transcription about 50 years ago 4. Research in this field has revealed that histone lysine acetylation modulates chromatin condensation and it is considered to be an epigenetic mark as part of the histone code 5. Although it took a long time until the discovery of tubulin as the first non-histone acetylation target 6, it is well established now that hundreds of eukaryotic proteins beyond histones can be acetylated and lysine acetylation has been recognized as a wide-spread PTM that may rival phosphorylation and ubiquitination in its prevalence 7-9. Interestingly, non-histone acetylated proteins can be signaling molecules in the cytoplasm, transcription factors in the nucleus, and metabolic enzymes in mitochondria, highlighting the significance of acetylation in regulating a plethora of cellular processes. 
The acetylation status of a protein depends on the coordinated and opposing function of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) which add and remove acetyl groups from proteins. The reversible acetylation of lysine, which involves neutralization of a positive charge 10, alters protein structure and it seems very likely to also alter enzymatic function in several cases 11-13. Focusing on KDACs, 18 proteins have been identified in the human and mouse genomes 14-16. Among them, mammalian sirtuins (also called class III histone lysine deacetylases) which are distinct from other members as they require NAD+ for their enzymatic function, have attracted extensive interest in this research field 16. In mammals, seven sirtuins (SIRT1-7) have been identified, each of them sharing a conserved 275-amino-acid catalytic core domain, which are mainly categorized according to their subcellular localization to the nucleus (SIRT1, 6, and 7), mitochondria (SIRT3, 4, and 5), or cytoplasm (SIRT2). SIRT1-3 have a robust deacetylation activity, while SIRT4 is reported to display ADP-ribosyltransferase activity, SIRT5 may function as a protein desuccinylase and demalonylase, and SIRT6 and SIRT7 display weak deacetylase activity but are involved in other types of acylations 17. In accordance with the significance of acetylation as a regulatory PTM modification involved in several cellular functions, sirtuins have also been implicated in a wide range of processes. After the first breakthrough studies establishing the role of sirtuins in life span extension, it has been shown that they are involved in diverse cellular functions including DNA repair, maintenance of genomic instability, apoptosis, response to stress and inflammation, control of energy efficiency, circadian clocks and metabolism, as well as contributing to the initiation and/or progression of age-related diseases such as cancer, neurodegeneration and type 2 diabetes 15,16.
Despite the significant progress in the field of sirtuin biology, more work remains to unravel undiscovered roles and functions through the identification of novel substrates. This is evidenced more emphatically by recent advances in high-resolution mass spectrometry (MS) based proteomics which have significantly increased the number of proteins found to be acetylated but most importantly have identified several different acetylated lysines in each protein, arguing that acetylation may be as wide-spread as other PTMs such as phosphorylation 7,8,17. Taking into consideration that specific deacetylases have not yet been identified for most of these acetylated proteins-substrates, it is reasonable to suggest that both in vitro and in vivo deacetylation assays are needed to confirm and establish an acetylated protein as a legitimate substrate of a specific deacetylase. In the experimental protocols described below, details will be given on how to perform both in vitro and in vivo deacetylation assays using SIRT2 as the specific deacetylase.

<table border="0" cellpadding="0" cellspacing="0" width="1543">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">cell culture dishes</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">T1110 and T1115</td>
			<td style="width:728px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">pCDH-puro-GFP lentiviral vector</td>
			<td style="width:239px;">System Biosciences</td>
			<td style="width:119px;">CD513B-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV- dR8.2 dvpr (packaging vector)</td>
			<td style="width:239px;">Addgene</td>
			<td>8455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV-VSV-G (envelope vector)</td>
			<td style="width:239px;">Addgene</td>
			<td align="right">8454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyethylenimine (PEI)&#160;</td>
			<td>Polysciences Inc.</td>
			<td align="right" style="width:119px;">24885</td>
			<td>other transfection reagents can be used as well. PEI is cost effective and very efficient in transfecting 293T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22&#956;m filters</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">F5512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polybrene</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H9268</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">fluorescent microscope</td>
			<td style="width:239px;">Carl Zeiss MicroImaging Inc.</td>
			<td style="width:119px;">Axiovert 200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:239px;">Invivogen</td>
			<td style="width:119px;">A11138-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:239px;">Corning</td>
			<td style="width:119px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag antibody</td>
			<td style="width:239px;">Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">M9272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">EGTA</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">34596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protease inhibitors coctail 100x</td>
			<td style="width:239px;">Biotool</td>
			<td style="width:119px;">B14001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichostatin A (TSA)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T8552</td>
			<td>selective inhibitor of class I and II histone deacetylases (HDACs) but not class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag agarose beads</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td style="width:239px;">Eppendorf</td>
			<td style="width:119px;">5417R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">rotator</td>
			<td style="width:239px;">Thermo Scientific</td>
			<td style="width:119px;">415220Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">filter tubes</td>
			<td style="width:239px;">Millipore</td>
			<td>UFC30HV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flag peptide&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td>F3290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivaspin Centrifugal Concentrator</td>
			<td>Sartorius Stedim Biotech S.A.</td>
			<td>VS0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SimplyBlue SafeStain solution&#160;</td>
			<td style="width:239px;">Invitrogen</td>
			<td style="width:119px;">LC6060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE LDS sample buffer (4x)</td>
			<td style="width:239px;">Life Techologies</td>
			<td style="width:119px;">NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Ac-K antibody</td>
			<td style="width:239px;">Cell Signaling</td>
			<td style="width:119px;">9441</td>
			<td>several anti-Ac-K antibodies are available. In our hands, the Cell Signaling antibody exhibits the highest sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl pH 7.5</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T5941</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NAD+</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">N0632</td>
			<td>required cofactor for sirtuins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nicotinamide (NAM)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">72340</td>
			<td>selective inhibitor class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pLKO.1 lentiviral vector&#160;</td>
			<td style="width:239px;">Addgene</td>
			<td style="width:119px;">8453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SIRT2 si RNA&#160;</td>
			<td style="width:239px;">Qiagen</td>
			<td style="width:119px;">GS22933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-SIRT2 antibody</td>
			<td style="width:239px;">Proteintech</td>
			<td style="width:119px;">15345-1-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bradford protein assay</td>
			<td style="width:239px;">BIO-RAD</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti Ac-K agarose beads</td>
			<td style="width:239px;">Immunechem</td>
			<td>ICP0388</td>
			<td></td>
		</tr>
	</tbody>
</table>

deacetylation assays to unravel the interplay between sirtuins (sirt2) and specific protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

Bir proteini için, in vitro ve in vivo deasetilasyonu deneylerde, her ikisi de deasetilasyon aktivitesi olan herhangi bir enzim için yasal bir deasetilasyon hedef olarak dikkate alınması gereken deasetilaz ve alt-tabaka arasındaki etkileşimi kurmak için yapılması gerekir. Deney yapılmadan daha önce in vitro olarak deasetilaz deneyi için, deasetilaz ve asetillenmiş protein substrata hem de saflaştırma gereklidir. Burada spesifik ...

Watch this Scientific Journal Video about Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates at JoVE.com

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Deasilasyon Tahliller Sirtuins arasında karşılıklı etkileşim (SIRT2) ve Özgül Protein yüzeylerde Unravel için

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

deacetylation-assays-to-unravel-interplay-between-sirtuins-sirt2

Research

JoVE Journal

Chemistry

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

8.2K Görüntüleme.  Feinberg School of Medicine, Northwestern University. This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Video: Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

Origami Inspired Desenli ve Reconfigurable Parçacıkların Self-assembly

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Kimya

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

Rapid Kolorimetrik Tahliller Nitelik Biyomoleküler Örnekleri RNA ve DNA ayırt etmek

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Gram-negatif bakterilerin izolasyonu ve Lipid A kimyasal karakterizasyonu

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Split-ve-havuz Sentezi ve Peptit Tersiyer Amid Kütüphanesi Karakterizasyonu

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. Isonicotinic acid, or its derivatives, are coupled to variable length diaminoalkane chains under an inert atmosphere in anhydrous DMF or DMSO with the use of a weak base, triethylamine, and a coupling agent, 1-propylphosphonic anhydride. The products precipitate from solution upon formation or can be precipitated by the addition of water. If desired, the ligands can be further purified by recrystallization from hot water. Dinuclear platinum complex synthesis using the bispyridine ligands is done in hot water using transplatin. The most informative of the chemical characterization techniques to determine the structure and gross purity of both the bispyridine ligands and the final platinum complexes is 1H NMR with particular analysis of the aromatic region of the spectra (7-9 ppm). The platinum complexes have potential application as anticancer agents and the synthesis method can be modified to produce trinuclear and other multinuclear complexes with different hydrogen bonding functionality in the bridging ligand.

This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin.

Çekirdekli Antikanser Maddeleri olarak Platinum bispiridin tabanlı Ligandların ve kompleks Sentezlenmesi için kullanılan amid bağlantı reaksiyon

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

Karakterizasyon, benzen polikarboksilik asitler kullanılarak Pirojenik karbon miktarının ve Bileşik özgü izotopik Analizi (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Bitki Doku Organik Asit belirlememize Kılcal Elektroforez kullanma: Bir Test Case incelenmesi<em&gt; Coffea arabica</em&gt; Tohumlar

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish between enantiomers (stereoisomers) of simple chiral species on the level of individual molecules. In this approach, a gaseous molecular jet of the sample expands into a vacuum chamber and intersects with femtosecond (fs) laser pulses. The high intensity of the pulses leads to fast multiple ionization, igniting a so-called Coulomb Explosion that produces several cationic (positively charged) fragments. An electrostatic field guides these cations onto time- and position-sensitive detectors. Similar to a time-of-flight mass spectrometer, the arrival time of each ion yields information on its mass. As a surplus, the electrostatic field is adjusted in a way that the emission direction and the kinetic energy after fragmentation lead to variations in the time-of-flight and in the impact position on the detector.
Each ion impact creates an electronic signal in the detector; this signal is treated by high-frequency electronics and recorded event by event by a computer. The registered data correspond to the impact times and positions. With these data, the energy and the emission direction of each fragment can be calculated. These values are related to structural properties of the molecule under investigation, i.e. to the bond lengths and relative positions of the atoms, allowing to determine molecule by molecule the handedness of simple chiral species and other isomeric features.

For small chiral species, Coulomb Explosion Imaging provides a new approach to determine the handedness of individual molecules.

Hipoglisemi arasında ayırt etmek için bir araç olarak Coulomb patlama görüntüleme

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

Raman ve IR spektroelektrokimyasal yöntemleri araçları olarak konjuge organik bileşikler çözümlemek için

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...