We thank the Australian Research Council (ARC) for ARC Future Fellowship (FT120100101) and ARC Centre of Excellence CE140100036) grants to P.T. and the Mark Wainwright Analytical Centre at UNSW for access to mass spectrometry and NMR facilities.

Not: Şekil 1 &#39;de gösterildiği gibi, aşağıdaki protokolü, bir protein-boya Bioconjugate sentezi için tasarlanmıştır notları yerleştirilmiş membran protein ile yardımcı olmak için durumlarda bu, serbest yüzey sistein ihtiva eden proteinlerle bir maleimid reaksiyonu için genel bir protokoldür. Bioconjugatlar protein polimer Bioconjugatlar ve sentetik protein dimeri (protein-protein) Bioconjugatlar. Bu özel durumda, protein izo-1 sitokrom C yüksek ölçüde özel bir işaretleme meydana sağlar tepkimeye uygun bir yüzey sistein artığı bulunur. ilgi konusu bir protein çoklu sisteyin artıkları ihtiva ediyor ise, aynı protokol özgüllük ve ürün homojenliği kaybı olsa geçerlidir. Özgüllük gerekli değilse N -hydroxysuccinimidyl esterleri ya da izotiosiyanatlar kullanılarak Kimya hedefleme yüzeyi lisin artıkları, daha basit bir yaklaşım olabilir. <img alt="Şekil 1" src="/files/ftp_upload/54157/54157fig1.jpg" /> <strong&gt; Şekil 1 Bioconjugation Reaksiyon Şeması. Örnek durumda, hafif hasat olarak rutenyum esaslı bir anteni molekülü (CYS102) rutenyum esaslı bir anteni molekül üzerinde bir asılı maleimid ve açık bir sistein kalıntısının Michael ilave edilerek sitokrom c bağlı olacaktır protein. Cyt c yüzeyinin kırmızı alan heme grubu gösterir. <a href="https://www.jove.com/files/ftp_upload/54157/54157fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Sitokrom c 1. Saflaştırma Not: Bu adım, tüm proteinlerin için geçerli değildir. Bununla birlikte, ticari bir kaynaktan elde edilen bir protein, bir daha arıtılmadan 13 çıkarılabilir gereken diğer istenmeyen protein izoformları içeren olduğunu bilmek önemlidir. <ol><li> Pr ultra saf su, 1 L (= 120 g mol-1 W E) sodyum dihidrojen fosfat 2.4 g çözülür20 mM NaH 2 PO 4 ihtiva eden bir tampon çözelti epare. pH 7-1 M NaOH ile pH ayarlayın. Not: Bu protokolde kullanılan fosfat tampon her gün taze olarak hazırlanmıştır, ve kullanımdan önce 0.2 um&#39;lik bir selüloz zar filtresi gerekir. </li><li> 20 mM NaH 2 PO 4 ve 1 M NaCl tampon yapmak için sodyum klorid 29.22 gramını (ağırlık M = 58.44 g mol-1), 20 mM NaH 2 PO 4 tampon, 500 ml içinde çözülür. Bu adım 1.6) &#39;de arıtma için elüsyon tamponudur. </li><li> PH 7 20 mM fosfat tamponu içinde 6 ml liyofilize sitokrom c (cyt c) 12.0 mg eritin. </li><li> Ayrıca, bir 1 M stok çözelti hazırlamak için ultra saf su 95,3 ul ditiyotreitol 14.7 mg (DTT, B, E = 154,25 g / Mol) çözülür. Not: bu madde, sulu çözelti içinde oksidatif deaktivasyon duyarlı olduğu gibi DTT stok çözeltisi taze hazırlanmalıdır. </li><li> ölmekProtein çözeltisi içine 1 M DTT çözeltisi 60 ul ette Cyt c azaltır. çözeltinin renk karıştırma üzerine ışık kırmızı koyu kırmızı değişecektir. </li><li> Herhangi bir kromatografik ortama enjekte etmeden önce 0.22 mikron düşük protein bağlayıcı PVDF şırınga filtresi aracılığıyla azaltılmış protein çözüm Filtre. </li><li> enjeksiyon döngüsü ve Hızlı protein sıvı kromatografi (FPLC) aletin UV-Vis detektörü ile 3.3 ml güçlü katyon değişim sütunu takın. </li><li> 1 ml / dk&#39;lık bir akış oranı kullanılarak, 20 mM pH 7 fosfat tamponunun 3 sütun hacim ardından ultra saf su, 3 kolon hacminde ile sütun dengelenmesi. </li><li> Yük 1 enjeksiyon döngü içine indirgenmiş ham cyt c ml ve 328 bir degrade yöntemi başlatmak - 5 sütun hacmi üzerinde 450 mM NaCl,. UV-Vis dedektör 280 nm ve 410 nm kanalı izlemek, ve en büyük zirve toplamak. </li><li> iso-2 sitokrom Zehir 1 M 2 kolon hacmi tuz konsantrasyonunu arttırmakE, C ve sütun boşaltılan sonra, aşağıdaki ham kısım enjekte edilmeden önce fosfat tampon 20 mM 2 sütun hacmi ile sütun yeniden dengeye. </li><li> tüm ham protein saflaştırılmış kadar tekrarlayın 1.9-1.10 adımları. </li><li> izo-1 ihtiva eden fraksiyonlar bir havuz ve 3.5 kDa moleküler ağırlık cutoff değeri (MWCO) sıkma filtre kullanılarak ve 3,000 x g de santrifüj bunları konsantre edin. </li><li> 3.5 kDa MWCO diyaliz kasetleri içine konsantre protein yükleyin ve su 2 değişikliklerle, ultra saf su bir gecede karşı dialyze. </li><li> 10 ul alan, aşırı saf su ile 100 ul&#39;ye seyreltilmesi, ve absorbans spektrumunun alınarak saf, konsantre protein çözeltisi konsantrasyonunu belirlemek. Düşük hacim kullanın, 100 ul kuvars küvet protein emme spektrumları elde etmek. 100 uM - Tipik olarak, seyreltilmemiş protein konsantrasyonu 50 mertebesindedir. <ol><li> Concentratio ölçmek için Cyt c karakteristik 410 nm tepe kullanınn 97.6 cm -1 mM -1 molar absorptivite değeri Beer-Lambert yasası kullanılarak: A = ε × c × ι Bir emme olduğu, ε molar emicilik, Cı mM konsantrasyonu, ve ι cm 13 küvetin yolu uzunluğudur. </li></ol></li><li> Bu noktada, 1 mL alikotlara protein bölmek ve gerekli olana kadar -20 ° C&#39;de dondurulmuş tutun. Sit c dondurulmuş ve yapı ya da fonksiyon kaybı olmadan çözülmüş, ancak tekrar döngüleri proteinin denatüre başlayacak edilebilir. Not: Proteinler farklı derecelerde dondurma tahammül. Örneğin, 5,9 dondurulamaz yeşil floresan protein yerine bir buzdolabı içinde depolanmıştır. </li></ol> Biyokonjugatlar c Sitokrom 2. Sentezi <ol><li> rutenyum 0.9 mg çözülür (II) bisterpyridine maleimide (Ru (II) (TPY) 2 -maleimid, 0.975 umol, 6 eşdeğer) ihtiva eden bir 600 ul. Not: kullanılan maleimid suda çözünür ise, su yerine asetonitril içinde bir stok çözeltisi hazırlanır. maleimid reaksiyon tamponu çözünür olması çok önemlidir. Dimetilsülfoksid, N, N-dimetilformamid ve asetonitril tüm yaygın düşük moleküler ağırlık 12, genellikle hidrofobik maleimid reaktiflerin çözünmesine yardımcı olmak için yardımcı maddeler kullanılmaktadır. </li><li> 20 mM&#39;ye seyreltilmiş EDTA çözünene kadar, tipik olarak (birkaç gram katı sodyum hidroksit ilave pH 7 olan 100 mM fosfat tamponu ve 100 mM etilendiamintetraasetik asit (EDTA, Mw = 292,24 g / mol) ihtiva eden bir tampon çözeltisi hazırlayın ) ile pH 7&#39;ye ayarlanır. </li><li> (286,65 g / Mol w = TCEP 15, m), aşırı saf su, 1 ml bir 10 mM stok çözelti hazırlamak için tris (2-karboksietil) fosfin hidroklorür 2.87 mg çözülür. Bu adım ithalatAnt protein sistein tam 15 bağlanması maleimide önceden indirgenmiş olduğundan emin olmak için. Not: bu madde, sulu çözelti içinde oksidatif deaktivasyon duyarlı olduğu TCEP stok çözeltisi, her bir reaksiyon için, taze hazırlanmalıdır. </li><li> 50 ml&#39;lik plastik bir tüp içinde 100 mM fosfat / EDTA stok çözeltisi 3 ml ultra saf su 11,4 ml birleştirin. Protein ve maleimid çözeltisi ile, daha da seyreltildi zaman tampon konsantrasyonunun 20 mM olacak. Not: etiketlendikten protein, bir trans membran proteini olup, uygun bir deterjan proteini çözünür hale getirmek için tampona eklenir sağlamak. bir deterjan kullanılmazsa zar proteini yavaş olumsuz reaksiyon verimlerini etki edecek ve, hızlandırabilir. Sonraki tüm saflaştırma aşamaları deterjan kullanın. </li><li> Fosfat-EDTA tampon maddesi ile arıtılmış, Cyt c 0.15 umol (50 uM çözelti 3 ml, 1 eşdeğer) eklenir. </li><li> TCEP stok SOLUT 7.5 ul ekleyinprotein çözeltisi iyon (0.075 umol, 0.5 eşdeğer) ve sistein oksidasyon nedeniyle dimerize olabilir herhangi bir protein azaltmak için 5 dakika boyunca karıştırıldıktan bırakın. Not: İlgili protein kolayca dimerize yoksa TCEP katmayın. indirgeyici olmayan koşullar altında, bir protein jeli çalıştırarak kontrol edin. </li><li> 24 saat süre ile, oda sıcaklığında, karanlıkta, Ru (II) (TPY) Cyt c düşük tamponlu solüsyona 2 -maleimid asetonitril çözeltisi 600 ul ekleyin ve reaksiyon karışımı, karıştırma bırakın. </li><li> 3.5 kDa MWCO döndürme filtreleri kullanarak reaksiyon karışımının, 3000 xg&#39;de santrifüj süzüntü berrak bitene kadar taze 20 mM fosfat tamponu ile 2-3 kez tekrar konsantre. arıtılmadan bir sonraki aşamada önce mümkün olduğu reaksiyon karışımından kadar tepkimemiş maleimid çıkarın. Not: Bu noktada ham bioconjugation Karışım, karanlıkta, buzdolabında saklanabilir. Centr ile reaksiyona girmeyen maleimid boya kaldırmak için önemlidirdepolamadan önce diyaliz ifuge maleimid yavaş olması ve spesifik olmayan protein yüzeyi üzerinde lizin kalıntıları ile reaksiyona girebilir. </li></ol> Sitokrom c Bioconjugatlar 3. saflaştırılması <ol><li> 20 mM NaH 2 PO 4 ve 0.5 M NaCl içeren bir tampon çözeltisi hazırlamak için, sodyum dihidrojen fosfat 2.4 g ve ultra saf su, 1 L sodyum klorür 29.22 g çözündürülür. Bu hareketsizleştirilmiş metal afinite kromatografisi (IMAC) saflaştırma için çalışan tampondur. </li><li> Imidazol 17 g (ağırlık M = 68,077 g mol-1), 20 mM NaH 2 PO 4 ve 0.5 M NaCl tampon 500 ml içinde çözülür. Bu IMAC arıtma için elüsyon tamponu olduğunu. </li><li> 1 M NaOH veya HCI kullanılarak pH 7&#39;ye Her iki tampon maddesi pH ayarlama, ve FPLC kullanılmadan önce 0.22 um rejenere selüloz membranlar yoluyla filtre. </li><li> Satın alınan IMAC sütun col yüklenen herhangi bir metal iyonları olmadan sevk edilir olarakUMN, ultra saf su 3 ultra saf su ml, 100 mM nikel asetat çözeltisi, 3 ml ve 6 ml ile yıkanması ile Ni2 + merkezli saflaştırılması için kolon hazırlanmıştır. Kolon hemen kullanılmak üzere değilse% 20 3 ml etanol ile yıkama ve bakteri büyümesini engellemek üzere buzdolabı içinde sütun saklayın. </li><li> Bir Ni 2+ enjeksiyon döngü ve UV-Vis dedektör arasındaki FPLC 1 ml IMAC sütunu -yüklü takın. </li><li> 20 mM fosfat, 3 kolon hacminde, 0.5 ml / dakika bir akış oranı ile 0.5 M NaCl tampon madde ile sütundan dengelenmesi. </li><li> Yük Ni2 + IMAC kolonu (0.22 um&#39;lik bir şırınga filtreden filtre) ham tepkime karışımından 100 ul. Rutenyum bisterpyridine-sitokrom C biokonjugatlannı elüte etmek için 125 mM 3 üzerinde sütun hacim 0 ilâ bir imidazol gradyanı, ardından reaksiyona girmemiş Cyt c elüt edilmesi için çalışan tampon, 3 kolon hacminde ile kolon yıkaması (Ru (II) &#39;-cyt c) . </li><li> C yıkamaTüm ham Bioconjugate saflaştırılmıştır kadar olumn 5 kolon hacmi için 250 mM imidazol tampon maddesi ile, daha sonra 20 mM fosfat, 0.5 M NaCl ve adımı tekrar 3.7 ile kolon yeniden dengeye. </li><li> Bioconjugate kesirler havuzu ve 3.000 x g santrifüj, 3.5 kDa MWCO döndürme filtresi kullanarak konsantre. </li><li> 3.5 kDa MWCO diyaliz kasetleri içine konsantre biokonjugatlannı yükleyin ve su 2 değişikliklerle, ultra saf su bir gecede karşı dialyze. </li><li> Izo-1 sitokrom c ile aynı molar absorptivitesi değerini kullanarak, UV-Vis saf, konsantre edildi Bioconjugate çözeltisinin konsantrasyonu belirlemek (97.6 mm-1 cm-1) 410 nm&#39;de. 100 mcM - Tipik olarak, Bioconjugate konsantrasyonu 50 mertebesinde olduğunu. </li><li> 25 ul kısma biokonjugatlannı bölün ve ihtiyaç duyulan kadar -20 ° C&#39;de dondurulmuş tutun. </li></ol> Sitokrom c Bioconjugatlar 4. Karakterizasyonu <ol><li> caydırmak-TOF MS tarafından Bioconjugate kütlesinin ermesi <ol><li> bir asetonitril / su / trifloroasetik asit çözeltisi, 1 ml kafeik asit, 10 mg çözülür (80: 20: 0.1, v / v / v). </li><li> kafeik asit çözeltisi 5 ul derişik protein çözeltisi 5 ul seyreltilir. </li><li> Nokta 0.5 MALDI hedef plaka üzerine kafeik asit çözeltisi ul ve çözüm kurumasını bekleyin. </li><li> Nokta 0.5 Bu kafeik asit yerinde üstündeki numune / matris çözümü ul, spot kurumasını bekleyin. &quot;Sandviç&quot; matris katmanları arasındaki numunenin bu üstündeki numune matris başka 0.5 ul nokta ve kurumasını bekleyin. </li><li> Proteinlerin 16 için uygun olan alet ayarları kullanılarak lineer şekilde kütle spektrumları elde edin. </li></ol></li><li> Jel elektroforezi ile biyokonjugatlar incelenmesi <ol><li> Her protein örneğinin 10 ul önceden karıştırılmış lityum dodesil sülfat (LDS, pH 8.4) tamponu (4x) t ile protein örneklerinin seyreltilmesi tarafından çalıştırılacak hazırlayıned oyuk başına yaklaşık 20 ug nihai konsantrasyon. indirgeyici koşullar gerektiren kuyuları, 500 mM ditiyotreitol (DTT) madde (10x) azaltmak için 1 ul ekle. </li><li> 10 dakika boyunca 70 ° C&#39;de ısı örnekleri. </li><li> Jel Yürütme tamponu hazırlamak için önceden karıştırılmış, 50 ml, 1 M MES, 1 M Tris baz,% 2 SDS, 20 mM EDTA (pH 7.7), ultra saf su 950 ml ticari bir kaynaktan tampon karışımı (20x) çalıştıran ekleyin. </li><li> jelden plastik tarak çıkarın ve jel çalışan tankında jel yerleştirin. Kuyular tamponu ile kaplıdır, böylece jel çalışan tampon tankı doldurun. </li><li> Numuneleri dikkatli bir şekilde prefabrik% 12 Bis-Tris üzerine, 1 mm boyunda, pipet uçları kullanılarak 10 oyuklu jel yükleme yardımcı olmak için. analiz yardım etmek için jel orta kuyuya prestained 10 protein moleküler ağırlık markerine (3-188 kDa) yükleyin. </li><li> 35 dakika için 200 V sabit gerilimde jel üzerinde çalıştırıldı. </li><li> 2 saat süre ile, ticari bir Coomassie mavisi solüsyonu ile jel leke ve ultrap yıkayın48 saat boyunca ure su. </li></ol></li><li> UV-Vis spektroskopisi yoluyla Bioconjugate saflık tayini <ol><li> Ru (II) (TPY) 2 -maleimid, izo-1 Cyt C ve Ru (II) &#39;-cyt Cı 5 uM çözeltilerinin 120 ul hazırlayın. </li><li> 250 nm 650 nm, tek ultra saf su içeren kuvars küvette bazal spektrumunu ölçün. </li><li> Küvet durulandı ve her bir ölçüm arasında kurutulur sağlanması her bir bileşenin spektrumunu ölçün. </li><li> Dalga boyunun bir fonksiyonu olarak her bir bileşenin absorbans Konu ve 1 belirlemek için nihai ürün ile başlangıç ​​malzemelerinin lineer bir miktar karşılaştırın Ru (II), (TPY) Cyt c 2 -maleimid 1 oranında reaksiyona. </li></ol></li></ol>

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Chem. 11 (28), 4602-4612 (2013).</li><li>Signor, L., Boeri Erba, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+Laser+Desorption/Ionization+Time+of+Flight+(MALDI-TOF)+Mass+Spectrometric+Analysis+of+Intact+Proteins+Larger+than+100+kDa.">Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa.</a> J. Vis. Exp. , e50635 (2013).</li><li>Foucher, M., Verdi&#232;re, J., Lederer, F., Slonimski, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+presence+of+a+non-trimethylated+iso-1+cytochrome+c+in+a+wild-type+strain+of+Saccharomyces+cerevisiae).">On the presence of a non-trimethylated iso-1 cytochrome c in a wild-type strain of Saccharomyces cerevisiae).</a> Eur. J. Biochem. 31, 139-143 (1972).</li><li>M&#252;ller, M., Azzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+labeling+of+beef+heart+cytochrome+oxidase+subunit+III+with+eosin-5-maleimide.">Selective labeling of beef heart cytochrome oxidase subunit III with eosin-5-maleimide.</a> FEBS Lett. 184 (1), 110-114 (1985).</li><li>Shen, B. Q., 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=Conjugation+site+modulates+the+in+vivo+stability+and+therapeutic+activity+of+antibody-drug+conjugates.">Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.</a> Nat. Biotechnol. 30 (2), 184-189 (2012).</li></ol>

The authors have nothing to disclose.

Bir bioconjugation önce başlangıç ​​malzemelerinin saflaştırılması büyük önem taşımaktadır. Ticari rekombinan kaynaklardan elde edilen proteinler, sıklıkla farklı yüzey kimya ve reaktiviteye sahip, ilgilenilen protein, diğer izoformları içerir. Örneğin, tarif edilen bioconjugation olarak, ticari olarak temin edilebilir Cyt Cı iki izo-1 ve izo-2 Cyt C 12,14,17 oluşan bir karışımı içerir. İzo-2 ve izo-1 sitokrom c formları temel fark, izo-1 sitokrom...

Bioconjugation kovalent bir boya, bir ilaç veya bir polimer gibi bir sentetik molekülü bir ya da sadece bir biyomolekülün bağlantı içerir. Protein bioconjugation yöntemleri artık yoğun protein yapma, floresan boya etiketleme 1,2 arasında değişen uygulamalarla birçok kimya, biyoloji ve nanoteknoloji araştırma gruplarında kullanılan (antikor) 3 (antikor ilaç konjugatları - ADK) -prodrugs protein dimerleri 4,5 sentezini , nanotıp 8 ve sistemler kimya 9 kullanılan kendinden montaj protein polimer melezler 6,7 kadar. kimya Özelliği, her zaman kritik olmasa da, hedef proteinin aktif site ile müdahale etmeyecek şekilde, en fonksiyonel proteini biyokonjugatlar için büyük önem taşımaktadır, bioconjugation kullanılır. Ilgilenilen protein i) hedefleme nadir veya benzersiz siteler: İdeal bioconjugation reaksiyonu da dahil olmak üzere, çeşitli kriterleri yerine getirmek gerekiyorii) iii) açılımı protein önlemek için denatüre edici olmayan koşullar altında devam, bu hedefe yönelik seçici olmak ve hedef protein genellikle alt milimolar konsantrasyonda kullanılabilir olarak iv) yüksek verimli olacak. Maleimid - sistein Michael katılması, tüm bu kriterlere uygun yakın geliyor ve bu nedenle uzun Bioconjugate Chemistry 10 alanında özel bir statüye iddia için vardır. i) kendi yüzeyi üzerinde tek bir sistein kalıntısı ihtiva eden bir çok protein, genetik doğru pH Reaksiyon sistein karşı yüksek oranda seçicidir ii) vardır tasarlanmış olabilir, çünkü bu, iii), sulu tamponlar içinde düzgün bir şekilde devam eder ve iv) çok hızlıdır bazı durumlarda, 11 5000 M-1 sn-1 aşan bildirilmiştir sistein içeren proteinlere maleimid ikinci dizi oranı sabiti ile. 12 ilgilenilen protein organik bir küçük (≈% 5-10) miktarı tolere edebilir sağlanan eş-çözücü, hemen hemen her maleimid fonksiyonlu boya, polymer, yüzey veya başka bir protein, protein ile bağlantılı olabilir. Buna ek olarak, maleimitler, yüksek pH diğer nükleofillerle reaksiyona daha yatkındır iyodoasetamid, daha proteinler üzerinde sistein daha özel olarak; ve disülfid değişim 13 önlemek için asidik pH değerinde tutulması gerekir disülfid tabanlı çekimleri daha kararlı. Burada, bir Ru (ll) tabanlı kromofor ve örnek olarak redoks proteini sitokrom c arasındaki reaksiyonu kullanarak tek bir sistein kalıntısı ihtiva eden bir proteine ​​maleimid işlevselleştirilmiş moleküllerin bağlanması için genel bir protokol sunulmuştur. Bu protokol, erişilebilir bir yüzeye sistein kalıntısı ve karşılık gelen maleimid-işlevselleştirilmiş bir hedef içeren, başka proteinler için de geçerlidir, bu bir protein, bir floresan boya, bir kromofor veya sentetik polimer.

<table border="0" cellpadding="0" cellspacing="0" width="913">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium dihydrogen phosphate</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">71496</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium hydroxide</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">71691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium chloride</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">73575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">cytochrome c, from saccaromyces cerevisiae</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">C2436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">dithiothreitol</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">TSKgel SP-5PW</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">Tosoh SP-5PW, 07161</td>
			<td>3.3 mL strong cation exchange column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15&#160;</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;">UFC900308</td>
			<td>3.5 kDa spin filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide-A-Lyzer mini dialysis units</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">66333</td>
			<td>3.5 kDa dialysis cassetes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ru(II) bisterpyridine maleimide</td>
			<td style="width:196px;">Lab made</td>
			<td style="width:151px;"></td>
			<td>see ref (14)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">acetonitrile</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">A3396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">ethylenediaminetetraacetic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">03609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tris(2-carboxyethyl)phosphine hydrochloride&#160;</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">93284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">imidazole</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">56749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">nickel acetate</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">244066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">AcroSep IMAC Hypercell column</td>
			<td style="width:196px;">Pall</td>
			<td style="width:151px;">via VWR: 569-1008</td>
			<td>1 mL IMAC column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.2 micron cellulose membrane filter</td>
			<td style="width:196px;">Whatman</td>
			<td style="width:151px;">Z697958</td>
			<td>47 mm filter for buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.2 micron PVDF membrane filter</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;">SLGV013SL</td>
			<td>syringe filters for proteins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">hydrochloric acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">84426</td>
			<td>extremely corrosive! Use caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">caffeic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">60018</td>
			<td>MALDI matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">trifluoroacetic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">91707</td>
			<td>extremely corrosive! Use caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SimplyBlue SafeStain</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">LC6060</td>
			<td>Coomassie blue solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE Novex 12% Bis-Tris Gel</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0342BOX</td>
			<td>precast protein gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">SeeBlue Plus2 Pre-stained Protein Standard</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">LC5925</td>
			<td>premade protein ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE LDS Sample Buffer (4X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0008</td>
			<td>premade gel sample buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE Sample Reducing Agent (10X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0004</td>
			<td>premade gel reducing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE MES SDS Running Buffer (20X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0002</td>
			<td>premade gel running buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Voyager DE STR MALDI reflectron TOF MS</td>
			<td style="width:196px;">Applied Biosystems</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Acta FPLC</td>
			<td style="width:196px;">GE</td>
			<td style="width:151px;"></td>
			<td>Fast Protein Liquid Chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Cary 50 Bio Spectrophotometer</td>
			<td style="width:196px;">Varian-Agilent</td>
			<td style="width:151px;"></td>
			<td>UV-Vis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Milli-Q ultrapure water dispenser</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;"></td>
			<td>ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Low volume UV-Vis Cuvette</td>
			<td style="width:196px;">Hellma</td>
			<td style="width:151px;">105-201-15-40</td>
			<td>100 microliter cuvette</td>
		</tr>
	</tbody>
</table>

synthesis of protein bioconjugates via cysteine-maleimide chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Biyokonjugatlar sentezi, üç ana yöntem ile teyit edilir: flight kütle spektrometrisi (MALDI-TOF MS), poliakrilamid jel elektroforezi Matris destekli lazer dezorpsiyon iyonizasyon süresi ve ultraviyole-Görünür (UV-Vis) spektroskopisi, Şekil 2&#39;de gösterildiği gibi, 3, 4. Ekteki küçük molekülün kütlesine karşılık gelen bir kütle artışı, ve reaksiyona girmemiş protein eksikliği Ru (II), başarılı bir şekilde ...

Watch this Scientific Journal Video about Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry at JoVE.com

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Bu protokol, reaktif saflaştırma, reaksiyon koşulları, Bioconjugate saflaştırma ve Bioconjugate karakterizasyonu dahil olmak üzere, bir maleimid proteini ihtiva eden bir sisteinin bioconjugation için gerekli olan önemli adımlar göstermektedir.

Protein Bioconjugatlar sentezi<em&gt; yoluyla</em&gt; Sistein-maleimid Kimya

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

synthesis-of-protein-bioconjugates-via-cysteine-maleimide-chemistry

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Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Chemical Applications of Statistical Analyses

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38.2K Görüntüleme.  The University of New South Wales.  Bu protokol, reaktif saflaştırma, reaksiyon koşulları, Bioconjugate saflaştırma ve Bioconjugate karakterizasyonu dahil olmak üzere, bir maleimid proteini ihtiva eden bir sisteinin bioconjugation için gerekli olan önemli adımlar göstermektedir. 

Video: Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides (CHIPS)

The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order to streamline synthesis by saving steps, time and material compared to conventional methods that require the introduction and removal of activating or directing groups. Therefore, the functionalization of C-H bonds is also attractive for green chemistry. Under oxidative conditions, two C-H bonds or one C-H and one heteroatom-H bond can be transformed to C-C and C-heteroatom bonds, respectively. Often these oxidative coupling reactions require synthetic oxidants, expensive catalysts or high temperatures. Here, we describe a two-step procedure to functionalize indole derivatives, more specifically tetrahydrocarbazoles, by C-H amination using only elemental oxygen as oxidant. The reaction uses the principle of C-H functionalization via Intermediate PeroxideS (CHIPS). In the first step, a hydroperoxide is generated oxidatively using visible light, a photosensitizer and elemental oxygen. In the second step, the N-nucleophile, an aniline, is introduced by Br&#248;nsted-acid catalyzed activation of the hydroperoxide leaving group. The products of the first and second step often precipitate and can be conveniently filtered off. The synthesis of a biologically active compound is shown.

Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides CHIPS

A two-step procedure for the synthesis of pharmaceutically active indole-derivatives by C-H functionalization with anilines is described, using photo- and Br&#248;nsted acid catalysis.

Intermediate Peroksitlerin yoluyla fotokimyasal ve Asit-katalize CH fonksiyonlandırmalar tarafından Antiviral tetrahidrokarbazole Türevlerinin Sentezi (CİPS)

The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order ...

Kimya

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Fonksiyonlu Sentezi ve Karakterizasyonu Metal-organik Altyapıları

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Synthesis and Reaction Chemistry of Nanosize Monosodium Titanate

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the material with Au(III) ions. The synthesis method was derived from a sol-gel process used to produce micron-sized monosodium titanate (MST), with several key modifications, including altering reagent concentrations, omitting a particle seed step, and introducing a non-ionic surfactant to facilitate control of particle formation and growth. The resultant nMST material exhibits spherical-shaped particle morphology with a monodisperse distribution of particle diameters in the range from 100 to 150&#160;nm. The nMST material was found to have a Brunauer-Emmett-Teller (BET) surface area of 285 m2g-1, which is more than an order of magnitude higher than the micron-sized MST. The isoelectric point of the nMST measured 3.34 pH units, which is a pH unit lower than that measured for the micron-size MST. The nMST material was found to serve as an effective ion exchanger under weakly acidic conditions for the preparation of an Au(III)-exchange nanotitanate. In addition, the formation of the corresponding peroxotitanate was demonstrated by reaction of the nMST with hydrogen peroxide.

The surfactant mediated sol-gel synthesis of nanosized monosodium titanate is described, along with preparation of the corresponding peroxide modified material. An ion-exchange reaction with Au(III) is also presented.

Nanoboyutlu Monosodyum Titanate Sentezi ve Reaksiyon Kimya

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the ...

The Synthesis of [Sn10(Si(SiMe3)3)4]2- Using a Metastable Sn(I) Halide Solution Synthesized via a Co-condensation Technique

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a sterically demanding ligand, has increased in recent years. The metastable Sn(I) halide is synthesized at&#160;&#34;outer space conditions&#34; via the preparative co-condensation technique. Thereby, the subhalide is synthesized in an oven at high temperatures, around 1,300 &#176;C, and at reduced pressure by the reaction of elemental tin with hydrogen halide gas (e.g., HCl). The subhalide (e.g., SnCl) is trapped within a matrix of an inert solvent, like toluene at -196 &#176;C. Heating the solid matrix to -78 &#176;C gives a metastable solution of the subhalide. The metastable subhalide solution is highly reactive but can be stored at -78 &#176;C for several weeks. On heating the solution to room temperature, a disproportionation reaction occurs, leading to elemental tin and the corresponding dihalide. By applying bulky ligands like Si(SiMe3)3, the intermediate metalloid cluster compounds can be trapped before complete disproportionation to elemental tin. Hence, the reaction of a metastable Sn(I)Cl solution with Li-Si(SiMe3)3 gives [Sn10(Si(SiMe3)3)4]2- 1 as black crystals in high yield. 1 is formed via a complex reaction sequence including salt metathesis, disproportionation, and degradation of larger clusters. Further, 1 can be analyzed by various methods like NMR or single crystal X-ray structure analysis.

The Synthesis of [Sn10SiSiMe334]2- Using a Metastable SnI Halide Solution Synthesized via a Co-condensation Technique

The disproportionation reaction of a metastable Sn(I) chloride solution, obtained via the preparative co-condensation technique, is used for the synthesis of a metalloid tin cluster compound.

sentezi [Sn<sub&gt; 10</sub&gt; (Si (SiMe<sub&gt; 3</sub&gt;)<sub&gt; 3</sub&gt;)<sub&gt; 4</sub&gt;]<sup&gt; 2</sup<sup&gt; -</sup&gt; Metastabil Sn (I) &#39;in, bir Co-kondensasyon yöntemi ile sentezlenmiş halojenür çözeltisinden kullanılması

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Bir 2&#39;-amino asit içeren RNA Oligomerlerinin Katı Faz Sentezi Protokolü<em&gt; Ç</em&gt; Tiyofenilmetil Değiştirme ve Sirküler Dikroizm ile Karakterizasyonlar

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Substrat bağlı Au Nanowires bir aktif yüzey büyüme mekanizması yoluyla sentezi

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

Otomatik Güncelleştirmeler, Pd ve Pt Aerogels üzerinden doğrudan çözüm tabanlı azaltma için hızlı sentez yöntemi

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

Esterleri bir yeşil Steglich esterleşme Asetonitril içinde yolu ile sentezi

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Escherichia coli-boş hücre Protein sentezi dayalı: sağlam, esnek ve erişilebilir platform teknolojisi için iletişim kuralları

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock copolymers are produced by one of three techniques: thin film rehydration, solvent switching or polymerization-induced self-assembly, and produce only flexible, polydisperse cylinders. Crystallization-driven self-assembly (CDSA) is a method which can produce cylinders with these properties, by stabilizing structures of a lower curvature due to the formation of a crystalline core. However, the living polymerization techniques by which most core-forming blocks are formed are not trivial processes and the CDSA process may yield unsatisfactory results if carried out incorrectly. Here, the synthesis of cylindrical nanoparticles from simple reagents is shown. The drying and purification of reagents prior to a ring-opening polymerization of &#949;-caprolactone catalyzed by diphenyl phosphate is described. This polymer is then chain extended by methyl methacrylate (MMA) followed by N,N-dimethyl acrylamide (DMA) using reversible addition&#8722;fragmentation chain-transfer (RAFT) polymerization, affording a triblock copolymer that can undergo CDSA in ethanol. The living CDSA process is outlined, the results of which yield cylindrical nanoparticles up to 500 nm in length and a length dispersity as low as 1.05. It is anticipated that these protocols will allow others to produce cylindrical nanostructures and elevate the field of CDSA in the future.

Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of &#949;-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined.

Biodegradable blok kopolimerlerin kristalizasyon Driven kendinden montajı ile Monodisperse silindirik Nanopartiküllerin sentezini

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock ...