Bu çalışma, bir NSF Kariyer Grant (MCB0954016) ve DOE Biyoenerji Araştırma Bursu (DEFG0208ER64694) tarafından desteklenmiştir.

1. Hücre kültürü (Şekil 1) <ol><li> Iz elementler, tuzlar, vitaminler ve yolağı soruşturma için en iyi olan, özellikle etiketli karbon substratları ile en az orta hücreleri büyütün. Hücre kültürü için sallayarak matara veya biyoreaktörler ya kullanın. Organik besinler, maya özü gibi, amino asit etiketleme ölçümü ile engel olabilir ve bu nedenle kültür ortamı mevcut olamaz. </li><li> UV / VIS spektrofotometre ile optimal bir dalga boyu (örneğin, Cyanothece 51.142 OD 730) kültür optik yoğunluk hücre büyümesini izleyin . </li><li> Hücreler ilk etiketli olmayan bir ortamda yetiştirilebilir. Orta-log büyüme aşamasında hücreleri etiketli orta aşılama (hacim aşılama oranı% 3 (v / v)) için kullanılacak tercih edilir. Etiketli ilk inokulum olmayan etiketli karbon giriş önlemek için etiketli aynı ortamda kültür (% 3 v / v aşılama oranı) alt kültür olmalıdır. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. Amino asit ekstraksiyon <ol><li> Santrifüj (10 dakika, 8000 × g) ile orta-log büyüme aşamasında Hasat alt-kültür hücreleri (10 ml). </li><li> 6M HCl 1.5mL içinde pelletini tekrar ve net bir cam, vida-üst GC flakon aktarmak. Şişeleri Cap ve biyokütle proteinler amino asitlere hidroliz 24 saat boyunca 100 ° C fırında koyun. Biyokütle pelet Hidroliz 20 ortak amino asitler (Şekil 2) 5 16 verim alınabilir. Sistein ve triptofan bozulmuş, glutamin ve asparajin sırasıyla, glutamat ve aspartat dönüştürülür. </li><li> 2 ml Eppendorf tüpler kullanılarak 5 dakika 20.000 amino asit çözeltisi × g Santrifüj, ve yeni bir GC şişeleri süpernatantlar aktarmak. Bu adım, hidroliz çözüm katı partikülleri. </li><li> GC flakon kapaklarını çıkarın ve örnekleri Thermo Scientific Reacti Vap evaporatör (not kullanarak hava akımı altında tamamen kuru bir dondurarak kurutma makinesi de kullanılabilir.o kuru örnekler). Bu adım, bir gecede yapılabilir. </li></ol> 3. Amino asit türevlendirme ve GC-MS koşulları GC ile amino asitler ya da yüksek ücret / polar metabolitler analizi, bu metabolitlerin amino asitler, uçucu ve gaz kromatografisi 2 ile ayrılabilir, böylece derivatized olmasını gerektirir. <ol><li> 150 mcL tetrahidrofuran (THF) ve N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide türevlendirme reaktifi 150 mcL ile kurutulmuş örnekleri çözülür. </li><li> Tüm örnekler, 65 ve 80 ° C &#39;de 1 saat süreyle arasında bir fırın veya bir su banyosunda inkübe edin. Flakonda metabolitleri çözülür emin olmak için ara sıra karıştırın. </li><li> 10 dakika 20.000 × g örnekleri santrifüj, ve ardından yeni bir GC şişeleri supernatant aktarmak. Süpernatant berrak ve sarımsı bir çözüm olmalıdır. Dedektörleri doygunluk nedeniyle, GC-MS ölçüm hassasiyeti yüksek conce etkilenebilirntration enjekte TBDMS derivatized amino asitler (bu örnekler çoğu zaman koyu kahverengi renk gösterir), bu nedenle, biz bu örneklerin, GC-MS ölçüm 6 önce THF kullanılarak seyreltik olmalıdır . </li><li> GC-MS (1:05 ​​ya da 1:10 split oranı, enjeksiyon hacmi = 1 mcL, taşıyıcı gaz helyum = 1.2 ml / dak). Numunelerin analiz Aşağıdaki GC sıcaklık programı kullanın: tutun 150 ° C, 2 dakika boyunca 3 artış ° C dakikada 280 ° C, 20 artış ° C dakikada 300 ° C ve daha sonra 5 dakika boyunca tutun. Solvent gecikme ~ 5 dakika (30 metre GC sütun için) olarak ayarlanabilir. MS oranı (m / z) şarj etmek için kitle aralığı 60 ve 500 arasında ayarlanabilir. </li></ol> 4. GC-MS veri analizi <ol><li> GC ayrılık izotop ayrımcılıktan TBDMS derivatized amino asit ölçümü de etkilenebilir. Işık izotoplar GC sütunundaki izotoplar kabarması daha hızlı hafifçe hareket. Potansiyel ölçüm önyargı azaltmak için, biz bütün tayf ortalamaamino asit pik 6 </li><li> TBDMS derivatized metabolitleri GC ve MS spektrumları 7 önce bildirilmiştir. Her amino asit için GC saklama süresi ve eşsiz m / z doruklarına Şekil 3&#39;te gösterilmiştir. </li><li> Amino asitler ya da merkez metabolitleri Türevlendirme 13 C (1.13%) de dahil olmak üzere önemli miktarda doğal etiketli izotoplar, 18 O (% 0,20), 29 Si (4.70%), ve 30 Si (3.09% ) tanıtır. Ham kitle isotopomer spektrumda doğal izotopları ölçüm gürültü yayınlanan yazılım 5, 8 kullanarak düzeltilebilir. Son izotopik etiketleme verileri kitle kesirler, örneğin, M 0, M 1, M 2, M 3 ve M 4 olarak bildirilmiştir (sıfır ile dört 13 C etiketli karbonlar içeren parçaları temsil eder). </li><li> 2-oxo-glutar: amino asit ölçümü sekiz önemli habercisi metabolitleri hakkında izotopik etiketleme bilgi sağlayabilir3-P-glycerate, asetil-KoA, erythrose-4-P, oksaloasetat, fosfoenolpiruvat, piruvat ve riboz-5-P, yedik. Bu metabolitlerin etiketleme desen birçok merkez metabolik yollar (Şekil 2) 9 tanımlamak için kullanılır. Etiketleme deneyler sonucu diğer biyokimya yöntemlerini kullanarak (örneğin, RT-PCR) teyit edilebilir. </li></ol> 5. Etiketli amino asit verileri kullanarak Pathway analizi Iyi tasarlanmış 13 C tracer deneyler sadece bir kaç temel amino asitler araştıran, tüm merkezi metabolizma gelişmiş 13 C-metabolik akı analizi yapılmadan birçok benzersiz yollar veya enzim faaliyetlerini ortaya çıkarabilir. <ol><li> Entner-Doudoroff yolu: [1 - 13 C] glukoz karbon kaynağı olarak kullanılabilir. Yolun etkinse, serin etiketleme, alanin 10 etiketleme anlamlı olarak daha düşük olacaktır . </li><li> Dallı TCA döngüsü: [1 - 13 C]pirüvat karbon kaynağı olarak kullanılabilir. TCA döngüsü kırık ise sadece bir karbon 11, 12 glutamat ile etiketlenmiş ise, aspartat iki karbonlar etiketli olabilir. </li><li> Mixotrophic metabolizmasında Calvin-Benson-Bassham döngüsü ile CO 2 fiksasyonu: CO 2 Sigara etiketli ve karbon substratlar etiketli karbon kaynağı olarak kullanılır. Calvin döngüsü fonksiyonel ise, serin ve histidin etiketleme diğer amino asitler ile karşılaştırıldığında önemli ölçüde, seyreltilmiş olacaktır. Bu yöntem orta 13 organik karbon kaynakları mevcut olduğunda göreli CO 2 fiksasyonu belirleyebilirsiniz. </li><li> Oksidatif pentoz fosfat: [1 - 13 C] glikoz, karbon kaynağı olarak kullanılabilir . Yolun etkinse, etiketli alanin&gt; 50% 12 olacaktır. </li><li> Anaplerotic yolağı (örneğin, KEP + CO 2 à oksaloasetat): 13 CO 2 ve olmayan etiketli karbon yüzeylerde (örneğin, gliserin veya piruvate) karbon kaynağı olarak kullanılabilir. Yolun etkinse, aspartat etiketleme, 13 serin alanin ve karşılaştırarak önemli ölçüde zenginleştirilmiş olacak. </li><li> Re-sitrat sentaz: [1 - 13 C] pirüvat karbon kaynağı olarak kullanılabilir. Enzim aktif ise β-karboksil grubu 3, 4, glutamat etiketlenmiştir. </li><li> Citramalate yolu: [1 - 13 C] piruvat, [2 - 13 C] gliserol, ya da [1 - 13 C] asetat, karbon kaynağı olarak kullanılabilir. Yolun etkinse, lösin ve izolösin etiketleme tutarları 14 aynıdır. </li><li> Serine isocitrate liaz döngüsü: [1 - 13 C] pirüvat veya [1 - 13 C] laktat karbon kaynağı olarak kullanılabilir. Yolun etkinse, serin üçüncü konuma karbon 15 etiketli olacaktır. </li><li> Besin kullanımı (örneğin, eksojen amino asitler): tam etiketli karbon yüzeyleri ve etiketli olmayan amino asitler ile bir kültür ortamıkullanılabilir. Hücrelerin bu takviye etiketli olmayan besinleri seçerek kullanır, biz biyokütle bu amino asitlerin önemli bir etiketleme seyreltme göreceksiniz. Bu yöntem, hangi besin takviyeleri 16 hücre tarafından tercih edilmektedir araştırmak için kullanılabilir. </li></ol> 6. Temsilcisi Sonuçlar Son biyoenerji çalışmaları, biyoenerji üretimi için yeni fototrofik mikroorganizmalar kullanılarak ilgi yeniden canlandı ve 2 yakalama CO . Geçmiş yıllarda, biyokimyasal bilgi merkezi metabolik yollar iyi değil, çünkü oldukça gelişmiş 13C-Metabolik Akı Analizler (13C-MFA) dahil olmak üzere birkaç 13C-metabolizma analizleri, merkezi metabolizmaları fototrofik bakteri araştırmak için uygulanan edilmiştir olmayan bu model organizmaların 10, 11, 17-20. Burada, biz, Cyanothece, alternatif bir izolösin yolun keşif 51.142 21 bir örnek sunuyoruz . Camgöbeğiothece 51.142 tipik izolösin sentez yolu 2-ketobutyrate treonin dönüşüm katalize enzimi (EC 4.3.1.19, treonin amonyak liaz) içermez. Izolösin yol gidermek için, 54 mM gliserol (2-13C,&gt;% 98) Cyanothece 51.142 ASP2 orta 22 (20 ml) büyür. Cyanothece 51.142 ana karbon kaynağı olarak etiketlenmiş gliserol 2. konumunu kullanır. Izolösin üç karbonlar ile etiketlenmiş ise treonin ve alanin bir karbon etiketli olduğunu gözlemliyoruz. Bu nedenle, çoğu organizmalar (Şekil 4) tarafından istihdam treonin güzergahtan Cyanothece 51.142 sentezi elde edilebilir. Öte yandan, lösin ve izolösin, belgeye dayanan aynı etiketleme kalıpları (M-15) ve parça (M-159) . Örneğin, M-15 isotopomer veri [] + (parçalanmamış amino asitleri içeren), lösin için aynı etiketleme (M0 = 0.01 = 0.21 M1 = 0.03, M2, M3 = 0.69) Ve izolösin (M0 = 0.01, M1 = 0.03, M2 = 0.24, M3 = 0,67). Böylece lösin ve izolösin aynı öncüleri (yani, piruvat ve asetil-CoA) sentezlenen olmalıdır. Bu gözlem, izolösin sentezi için citramalate yol etiketli karbon geçiş ile tutarlıdır. Bu yolun onaylamak için, Joint Genom Enstitüsü veritabanında arama ve Cyanothece citramalate sentaz Cima (cce_0248) varlığı bulmak. <img src="/files/ftp_upload/3583/3583fig1.jpg" alt="Şekil 1"/> Şekil 1: 13 C-destekli yolak analizi adımları . <img src="/files/ftp_upload/3583/3583fig2.jpg" alt="Şekil 2"/> Şekil 2 metabolik öncüleri etiketleme desen elde etmek için kullanılan Amino asitler. ACoA, asetil-CoA; AKG, α-Ketoglutarate; C5P, riboz 5-fosfat; CIT, sitrat; E4P, erythrose 4-fosfat; G6P, glukoz 6-fosfat; OAA<html> oksaloasetat; KEP fosfoenolpiruvat; PGA, 3-fosfogliserat; PYR, piruvat. <img src="/files/ftp_upload/3583/3583fig3.jpg" alt="Şekil 3"/> Şekil 3. 16 amino asitler için GC zirveleri. MS TBDMS derivatized amino asitler iki parça halinde kırık: (E-57) +, tüm amino asit içeren ve (M-159) +, α amino asidin karboksil grubu yoksun. Lösin ve izolösin için (E-57) + diğer kitle zirveleri ile çakışmış oldu. Biz tüm amino asit etiketleme analiz parçası (E-15) + kullanmanızı öneririz. (F302) + grup, bir amino asit omurga sadece ilk (α-karboksil grubu) ve ikinci karbonlar içeren, en çok amino asit tespit edilmiştir. Bu MS tepe sık sık yüksek gürültü-sinyal oranları olduğundan, (F302) + metabolik akıları 7 kantitatif analiz için tavsiye değildir. igure 4 &quot;/&gt; Şekil 4 Cyanothece 51.142 (önceki kağıt değiştirilmiş) 21 izolösin yollar geçişler Etiketleme.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariability+of+central+metabolic+flux+distribution+in+Shewanella+oneidensis+MR-1+under+environmental+or+genetic+perturbations.">Invariability of central metabolic flux distribution in Shewanella oneidensis MR-1 under environmental or genetic perturbations.</a> Biotechnol Prog. 25, 1254-1259 (2009).</li><li>Zamboni, N., Fendt, S. M., Ruhl, M., Sauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C-based+metabolic+flux+analysis.">13C-based metabolic flux analysis.</a> Nature Protocols. 4, 878-892 (2009).</li></ol>

Çıkar çatışması ilan etti.

Bu protokol etiketli substrat hücre GC-MS ile amino asitler beslenme ve elde edilen izotopik etiketleme desen ölçme oluşmaktadır. MS veri (m / z oranlarına) MS iyonlarının etiketleme sadece toplam miktar vermek zamandan beri, biz inceleyerek hem de parçalanmamış (E-57) + m / z oranları ve parçalanmış amino asitler, amino asitlerin isotopomer dağılımları değerlendirmek için (yani, (M-159) + (F302) +). Ayrıca, kimyasal olarak benzer bir ortamı ancak farklı etiketlem...

<table border="1"><tbody><tr><th> Reaktifi Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> TBDMS </td><td> Sigma-Aldrich </td><td> 19915 </td><td> - </td></tr><tr><td> THF </td><td> Sigma-Aldrich </td><td> 34865 </td><td> - </td></tr><tr><td> Etiketli karbon substrat </td><td> Cambridge İzotop Laboratuvarlarında </td><td> Deneysel gereksinimi bağlıdır. </td><td> Web sitesi <a href="http://www.isotope.com">:</a> http://www.isotope.com </td></tr><tr><td> Gaz kromatograf </td><td> Agilent Technologies </td><td> Hewlett-Packard, model 7890A </td><td> - </td></tr><tr><td> GC Kolonları </td><td> J &amp; W Scientific, Folsom, CA </td><td> DB5 (30m) </td><td> - </td></tr><tr><td> Kütle spektrometresi </td><td> Agilent Technologies </td><td> 5975C </td><td> - </td></tr><tr><td> Reacti-Vap Evaporator </td><td> Thermo Scientific </td><td> TS-18.825 </td><td> Amino asit örnekleri kurutma için </td></tr></tbody></table>

metabolic pathway confirmation and discovery through 13c-labeling of proteinogenic amino acids

Mikroplar, biyokimya ve işlevsel genomik yöntemleri kullanılarak araştırılmalıdır karmaşık metabolik yollar var. Hücre merkezi metabolizma incelemek ve yeni enzimler keşfetmek için önemli bir teknik, 13 C-destekli metabolizma analizi 1 . Bu teknik izotopik etiketleme, mikropların 13 C etiketli substratları ile beslenen sayede dayanmaktadır. Biyokimyasal ağ metabolitleri arasında atom geçiş yolları takip ederek, işlevsel yollar belirlemek ve yeni enzimler keşfetmek. Transkriptomik ve proteomik, metabolik yollarının isotopomer yardımlı analizi için tamamlayıcı bir yöntem yaklaşımları üç önemli adımlar 2 içeren İlk 13 C etiketli substratlar hücreleri büyür. Bu adımda, orta ve etiketli yüzeylerin seçimi kompozisyon iki önemli faktör vardır. Besin takviyeleri etiketli karbon ölçümü gürültüleri önlemek için, Tek bir karbon kaynağı ile en az bir ortam gereklidir. Ayrıca, etiketli bir substrat seçim analiz edilen yolun açıklamak ne kadar etkili dayanmaktadır. Roman enzimler genellikle farklı reaksiyon stereokimya genel veya ara ürünler, içerdiğinden, tek başına etiketli karbon yüzeyler, roman yollar 3, 4 tespiti için düzgün etiketli olanlardan daha roman yollarının tespiti için daha bilgilendirici İkincisi, biz GC kullanarak amino asit etiketleme kalıplarını analiz -MS. Amino asitler protein bol miktarda bulunmaktadır ve dolayısıyla, biyokütle hidroliz elde edilebilir. Amino asitler, GC ayırma önce N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) derivatized olabilir. TBDMS derivatized amino asitler, MS ve parçaları farklı diziler sonucu parçalanmış olabilir. Parçalanmış ve parçalanmamış amino asitler (m / z) oranı sorumlu kitle dayanarak, merkezi metabolitlerin mümkün etiketli desen anlayamayacağımızamino asitlerin öncüleri Üçüncü olarak, biz bu yolları 2 aktif olup olmadığını onaylamak, isotopomer verilere göre, önerilen yollar 13C karbon geçişleri izlemek ve. Amino asit ölçümü izotopik etiketleme merkezi metabolizma sekiz önemli habercisi metabolitleri hakkında bilgi sağlar. Bu metabolik anahtar düğümler ilişkili merkezi yollarının fonksiyonlarını yansıtır. Aminoasittir ile 13 C-destekli metabolizma analizi kötü karakterize mikrobiyal metabolizma 1 fonksiyonel karakterizasyonu için yaygın olarak kullanılır . Bu protokol, yeni enzimatik fonksiyonları keşfetmek için etiketli karbon substratların kullanımını göstermek için model suşu olarak Cyanothece 51.142 kullanacaktır.

Watch this Scientific Journal Video about Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids at JoVE.com

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13 C-izotop etiketleme çeşitli türleri için mikroorganizmaların hücre merkezi metabolizma belirlenmesi için yararlı bir tekniktir. Hücreleri belirli bir substrat ile etiketli kültüre edildikten sonra, GC-MS ölçüm aminoasittir benzersiz etiketleme desen dayalı işlevsel metabolik yollar ortaya çıkarabilir.

Sayesinde Metabolik Pathway Onay ve Keşif 13</supAminoasittir> C-etiketleme

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

metabolic-pathway-confirmation-discovery-through-13c-labeling

Research

JoVE Journal

Biology

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

24.1K Görüntüleme.  Washington University. 13 C-izotop etiketleme çeşitli türleri için mikroorganizmaların hücre merkezi metabolizma belirlenmesi için yararlı bir tekniktir. Hücreleri belirli bir substrat ile etiketli kültüre edildikten sonra, GC-MS ölçüm aminoasittir benzersiz etiketleme desen dayalı işlevsel metabolik yollar ortaya çıkarabilir.

Video: Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Biyoloji

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

Bir adım metabolomik: Karbonhidratlar, Tek Bir Usul Sayısallaştırılmış Organik ve Amino Asitler

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Nükleik Asitlerin NanoDrop Microvolume kantitasyonu

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Zebra balığı Embriyolar Metabolik Profil Analizi

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolik Oranı Ölçümü<em&gt; Drosophila</em&gt; Respirometrik kullanarak

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Büyüme-tabanlı Belirlenmesi ve Protein Parçalanabilirliklerinin Genetik Talepler biyokimyasal Onay<em&gt; Saccharomyces cerevisiae</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

Aracılığıyla DEĞİŞTİR Protein-protein etkileşimleri İnhibitörlerinin Geliştirilmesi: Tasarım ve Geliştirme Olmayan ATP Rekabetçi CDK inhibitörleri Uygulama

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

Bir Kullanarak Modeli Proteinler içine Kuralsız Amino Asit kalıntı özgü Ortaklığın<em&gt; Escherichia coli</em&gt; Hücre içermeyen Transkripsiyon-çeviri Sistemi

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Nükleik Asitlerin süzülmesi İzolasyonu: basit ve hızlı DNA izolasyonu,

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Sayesinde Metabolik Pathway Onay ve Keşif 13 C-etiketleme

Sayesinde Metabolik Pathway Onay ve Keşif^{13 C-etiketleme}