Bu çalışma, NSF ödül # 0920274 ve Güney Carolina Deney İstasyonu Projesi (SC-1.700.340) KSS tarafından desteklenen oldu. Bu makale, Clemson Üniversitesi Experiment Station 5929 Teknik Katkı No.

Bu protokolün genel şeması Şekil 1&#39;de özetlenmiştir. 1. Standart Eğriler ve Tahliller Çözüm Hazırlığı <ol><li> Hidroksilamin-HCl 2 mol / L çözelti 100 mL hazırlayın. Hidroksilamin hidroklorür 13,9 g (MW 69.49 g / mol) tartılır ve yaklaşık 50 ml distile deiyonize su (GKD 2 O) içinde çözülür. Potasyum hidroksit pelet veya konsantre bir çözüm kullanarak 7.0 pH ayarlayın. Son hacim 100 mL &#39;ye getirin. Çözüm 30 gün kadar ya da 4 ° C&#39;de 90 gün öncesine kadar oda sıcaklığında saklanabilir. </li><li> 100 mL demir klorür / hidroklorik asit solüsyonu hazırlayın. 13.5 gr ferrik klorür (MW 270,32 g / mol) tartılır ve yaklaşık 50 ml GKD 2 O içinde çözülür 41.3 ml konsantre hidroklorik asit (12.1 mol / L) ekleyin ve bir son hacim 100 mL getirmek. Ferrik klorür son konsantrasyonu 0,5 mol / L ve hidroklorik asit konsantrasyonu son 5 mol olacak olacak/ L. Çözüm oda sıcaklığında saklanır. </li><li> Trichoroacetic asit 1.84 mol / L çözelti 100 ml hazırlayın. Trikloroasetik asit (MW 163,39 g / mol) 30.0 gr tartılır, GKD 2 O içinde çözülür ve son hacim 100 mL getirmek. Çözüm oda sıcaklığında saklanır. </li><li> 5 ml 0.1 mol / L asetil fosfat çözüm hazırlayın. Tartılır, 0,092 gr asetil fosfat (MW 184,06 g / mol) GKD 2 O içinde çözülür ve son hacim 5 ml getirmek. Kısım mikrosantrifüj tüpler (tüp başına 0.25 ml) çözüm ve kullanıma kadar -20 ° C&#39;de dondurma. Çözülmüş bir çözüm buz konulmalıdır ve yaklaşık 1 saat için de kullanılabilir. Çözülmüş oldu Asetil fosfat çözüm refrozen ve yeniden kullanılan olmamalıdır. </li><li> ADP 0.1 mol / L çözelti 5 ml hazırlayın. ADP 0.24 g (MW 472,5 g / mol) tartılır, GKD 2 O içinde çözülür ve son hacim 5 ml getirmek. -20 Kısım mikrosantrifüj tüpler çözeltisi (tüp başına 0,5 ml) ve donma ° C &#39;ye kadarkullanın. Kullanana kadar buz çözülmüş çözüm tutun. </li><li> Tris, 1 mol / L çözelti 50 mL hazırlayın. 6.06 g Tris (MW 121,14 g / mol) tartılır ve 40 ml GKD 2 O. içinde çözülür Çözeltinin pH 7.0 hidroklorik asit kullanarak ayarlayın ve son hacim 50 ml getirmek. Oda sıcaklığında saklayın çözüm. </li><li> 10 ml, 1 mol / L magnezyum klorür solüsyonu hazırlayın. 2.03 gr magnezyum klorür (MW 203,31 g / mol) tartılır, GKD 2 O içinde çözülür ve son hacim 10 ml getirmek. Çözüm oda sıcaklığında saklayın. </li><li> Ferrik klorür / hidroklorik asit çözeltisi ve trikloroasetik asit çözeltisi eşit hacimlerde karıştırılması ile 25 ml geliştirme çözümü hazırlayın. Geliştirme çözümü, her gün taze olarak hazırlanır ve oda sıcaklığında muhafaza edilmelidir. </li></ol> 2. Asetil Fosfat Standart Eğrisi hazırlanması <ol><li> Asetil fosfat standart eğri bilinen miktarda asetil fosfat kullanılarak oluşturulur. Ha altı standart eğrit noktaları uygundur. Standartları 0.03 μmoles 0.9 μmoles son 0,1-3 mmol / L konsantrasyonları ile ilişkilidir başına 300 mcL, aralık 300 mcL son hacim 100 mM Tris çözüm uygun konsantrasyonlarda asetil fosfat stok solüsyonu sulandırınız. Asetil fosfat içermeyen bir kontrol örneği de hazırlıklı olmalısınız. </li><li> Her standart ve kontrol için 37 ° C ısı bloğu 1 dakika inkübe edin. </li><li> Her bir standart hidroksilamin hidroklorür çözüm ve üç kez sallayarak kontrolü ve mix 50 mcL ekleyin. Standart 60 ° C ısı bloğu içine yerleştirin ve beş dakika boyunca inkübe edin. </li><li> 100 standartlarına geliştirme çözümü mcL ve kontrol, üç kez sallayarak karıştırmak ekleyin ve renk oda sıcaklığında en az bir dakika geliştirmek için olanak sağlar. </li><li> 1 dakika 18.000 xg mikrosantrifüj tüm standartları ve kontrol santrifüjleyin. </li><li> Spektrofotometrik bir örnekleri ölçünspektrofotometri için boş olarak kumanda kullanılarak, 1 ml plastik küvetler kullanılarak t 540 nm. </li><li> Uygun veri analizi ve grafik yazılımı (örn. Kaleidagraph, Sinerji Yazılım) kullanarak standart bir eğri oluşturur. R 2 değerleri 0.99 ya da daha fazla arzu edilir. Veri asetil fosfat mevcut μmoles karşı 540 nm&#39;de absorbans olarak çizilir. </li></ol> 3. Asetat Kinaz Aktivitesi için Testi <ol><li> Açıklanan çözümlerini kullanarak, nihai konsantrasyonu 0,1 mol / L Tris tampon, 10 mmol / L magnezyum klorür, 5 mmol / L ADP ve 2 mmol / L asetil fosfat içeren bir reaksiyon karışımı hazırlayın. 10 ml reaksiyon karışımı 8.2 ml GKD 2 O, 1.0 ml Tris çözüm, 0.1 ml magnezyum klorür çözeltisi, 0.5 mL ADP çözüm, ve 0.2 mL asetil fosfat çözümü gerekecektir. Mikrosantrifüj tüpleri ile 300 ul alikotları reaksiyon karışımı dağıtın. Kinetik parametreleri ve optimizasyon substrat konsantrasyonlarının belirlenmesi için, bir substratçeşitli ve diğer reaksiyonlarda bir sabit konsantrasyonda düzenlenen. </li><li> 37 az 1 dakika süreyle reaksiyonlar Ön-inkübe ° C ısı bloğu. </li><li> İstediğiniz bir reaksiyon enzim miktarını ekleyin ve tüp 37 ° C ısı bloğu hemen dönün. Ayrıca, herhangi bir enzim eklenir kontrol reaksiyon içerir. Çeşitli reaksiyonlar yapılır ise, belirli zaman aralıklarında tüplerine enzim ekleyin. Zaman takip etmek için emin olun. (NOT: kullanılacak enzim miktarı asetil fosfat veya ADP substrat tükenmiş olmayacaktır sağlamak için optimize edilmiş olması gerekecektir - Tartışma bölümüne bakın) </li><li> 5 dakika sonra, her bir reaksiyon ve kontrolü, mix hidroksilamin hidroklorür çözüm 50μl ekleyin, ve tüpler 60 ° C ısı bloğu. Çeşitli reaksiyonlar yapılmaktadır enzim eklendi, bu aynı zaman aralığında olmalıdır. </li><li> 5 dakika reaksiyonlar ve kontrol inkübe, 60 ° C </li><li> 100 &amp; ekleyinizmu, her tepki ve kontrol laboratuarlarında tüp rafa, mix, yer ve geliştirme çözümü L. Renk, en az 1 dakika geliştirmek. Sağlayın </li><li> 1 dakika 18.000 xg mikrosantrifüj bütün tüpler santrifüjleyin. </li><li> Ölçü örnekleri 1mL plastik küvetler kullanılarak 540 nm&#39;de spektrofotometrik ve asetil fosfat standart eğri ile karşılaştırıldığında, asetil fosfat mevcut miktarını belirlemek. </li><li> Tükenmiş asetil fosfat substratı miktar kontrolü enzim eksikliği bulunan asetil fosfat miktarı enzimatik reaksiyon sonra kalan asetil fosfat miktarı çıkarılarak tespit edilebilir. Uygun veri ve grafik yazılımı (örn. Kaleidagraph, Sinerji Yazılım) kullanarak veri analizi yapın. R 2 değerleri 0.97 &#39;den daha fazla arzu edilir. </li></ol> 4. Temsilcisi Sonuçlar: Bu testin amacı, doğrudan asetat kinaz aktivitesini ölçmek içinasetat oluşumu iyon. Bu asetat üretimi için hiçbir kolay, doğrudan ölçüm, asetil fosfat substratı tüketimi ölçerek yapılır. Asetil fosfat enzimatik reaksiyon sırasında belirli bir zaman sonra kalan asetil hydroxamate ve daha sonra 540 nm dalga boyunda ölçülür (Şekil 2) kırmızımsı bir renk olan ferrik hydroxamate kompleksi, dönüştürülür. Standartlarda μmoles asetil fosfat mevcut komplo karşı absorbans standart eğri Her reaksiyon kalan asetil fosfat miktarını belirlemek için kullanılır. Standart eğri sıfır noktası üzerinden doğrusal olmalıdır. Şekil 3&#39;te gösterildiği bir temsilci standart eğrisi verileri de lineer denklem uygun belirten μmole asetil fosfat bugünü ve R 2 değeri 0.99 başına 1,2332 absorbans birimi bir eğime sahiptir. Lineer oturması için standart eğri veri denklemi reaksiyon kalan asetil fosfat miktarını hesaplamak için kullanılır. Μmolestüketilen asetil fosfat kontrol reaksiyon enzim ve enzimatik reaksiyon sonra kalan asetil fosfat μmoles yoksun bulunan asetil fosfat μmoles arasındaki fark olarak belirlenir. Saflaştırılmış, rekombinant M. thermophila asetat kinaz açıklanan protokolü kullanılarak analiz edildi. Şekil 4&#39;te gösterildiği deney için 5 mmol / L, ADP konsantrasyonu sabit tutulur ve reaksiyon asetil fosfat konsantrasyonu asetil fosfat için K m belirlemek için farklı oldu. Beklendiği gibi, bu deney her substrat enzim için standart Michaelis-Menten gibi kinetiği takip eder ve hiperbolik bir doyma eğrisi asetil fosfat konsantrasyonu karşı komplo faaliyet göstermektedir. Asetil fosfat için görünür K m değeri 0.27 ± 0.47 yayınlanan K m değeri ile karşılaştırıldığında olumlu 0.01 mmol / L ± akuple olarak kullanarak 0.01 mmol / L13 söylüyorlar. Doğrudan analiz aynı zamanda, standart birleştiğinde tahlil kullanarak mümkün değildir ATP daha başka yüzeylerde kullanmak asetat kinaz aktivitesini ölçmek için kullanılabilir. Rekombinant E. Saf asetat oluşturan yönde PP i yerine ATP üretir histolytica asetat kinaz 16, sodyum fosfat ADP yerine kullanarak bu testte maruz kaldı. Reaksiyon sodyum fosfat konsantrasyonu 0.2 mol / L olarak sabit tutuldu ve asetil fosfat konsantrasyonu çeşitlilik oldu. M. thermophila asetat kinaz, E. histolytica asetat kinaz asetil fosfat için Michaelis-Menten gibi kinetiği takip ve hiperbolik bir doygunluk eğri (Şekil 5) gözlendi. Asetil fosfat için görünür K m değeri 0.50 ± 0.006 mmol / L Reeves ve Guthrie 15 doğal enzim asetil fosfat için 0,06 mmol / L K m değeri tespit;<html> hiç enzim sadece kısmen saflaştırılmış ve tahlil da sadece kısmen arıtılmış dört kaplin enzimler dahil oldu. Bu nedenle, doğrudan bu değerleri karşılaştırmak zordur. Protokol Programı <ol><li> Asetil fosfat standart eğri oluşturma </li><li> Istenen konsantrasyona enzim sulandırınız </li><li> Mikrosantrifüj tüpler için reaksiyon karışımı ve kısım 300 ul hazırlayın </li><li> 1 dakika için 37 Preincubate reaksiyonlar ° C </li><li> Reaksiyonlar, belirli zaman aralıklarında enzim ekleyerek başlatın </li><li> Reaksiyonları durdurmak ve 60 yaşında inkübe aynı aralıklarla hidroksilamin ° C 5 dakika </li><li> Ferrik klorür / renk gelişimi için trikloroasetik asit çözeltisi ekle </li><li> 1 dakika 18.000 xg tüpler Santrifüj </li><li> 540 nm dalga boyunda absorbansı ölçülür </li><li> Standart eğrisine göre tüketilen asetil fosfat miktarını belirlemek. </li></ol>tp_upload/3474/3474fig1.jpg &quot;/&gt; Şekil 1: Protokol Programı <img alt="Şekil 2" src="/files/ftp_upload/3474/3474fig2.jpg" /> Şekil 2: Asetil fosfat standartları . Artan konsantrasyonlarda asetil fosfat ile reaksiyona açıklandığı gibi hidroksilamin ve renk geliştirilmiştir. Asetil fosfat eksik kontrolü, parlak sarı, ve asetil fosfat artan miktarda içeren reaksiyonlar gittikçe koyu renkli. Bu renk değişimi, 540 nm dalga boyunda ölçülür. <img alt="Şekil 3" src="/files/ftp_upload/3474/3474fig3.jpg" /> Şekil 3. Örnek asetil fosfat standart eğri. Asetil fosfat değişen miktarlarda hidroksilaminle tepki ve renk geliştirme çözümü ve absorbans 540 nm&#39;de ölçüldü. Μmoles asetil fosfat karşı 540 nm&#39;de absorbans çizilen ve veri doğrusal bir uyum uygulanmıştır./ P&gt; <img alt="Şekil 4" src="/files/ftp_upload/3474/3474fig4.jpg" /> Şekil 4 ATP üreten M. asetil fosfat kullanımı thermophila asetat kinaz. Asetil fosfat değişen konsantrasyonları ile 5 mmol / L ADP varlığı asetat oluşumu yönünde enzimatik aktivite tespit edilmiştir. Asetil fosfat tüketilen miktar tepki asetil fosfat başlangıç ​​tutarı ve enzimatik reaksiyon sonra kalan asetil fosfat miktarı arasındaki fark olarak tespit edildi. Tahliller gösterilen hata barları ile üç nüsha halinde yapılmıştır. <img alt="Şekil 5" src="/files/ftp_upload/3474/3474fig5.jpg" /> Şekil 5 PP asetil fosfat kullanımı i üreten E. histolytica asetat kinaz. Asetat oluşumu yönünde enzimatik aktivite 0 varlığı tespit edildi.Asetil fosfat konsantrasyonları değişen 2 mol / L sodyum fosfat. Asetil fosfat tüketilen miktar tepki asetil fosfat başlangıç ​​tutarı ve enzimatik reaksiyon sonra kalan asetil fosfat miktarı arasındaki fark olarak tespit edildi. Tahliller gösterilen hata barları ile üç nüsha halinde yapılmıştır.

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+enzymes+involved+in+the+proponic+acid+fermentation.">Purification and properties of enzymes involved in the proponic acid fermentation.</a> Journal of Bacteriology. 87, 171-187 (1964).</li><li>Clarke, P. M., Payton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+assay+for+acetate+in+spent+bacterial+culture+supernatants.">An enzymatic assay for acetate in spent bacterial culture supernatants.</a> Anal. Biochem. 130, 402-405 (1983).</li><li>Mukhopadhyay, S., Hasson, M. S., Sanders, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+assay+of+acetate+kinase+activity:+measurement+of+inorganic+phosphate+release+generated+by+hydroxylaminolysis+of+acetyl+phosphate.">A continuous assay of acetate kinase activity: measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate.</a> Bioorg. Chem. 36, 65-69 (2008).</li><li>Nakajima, H., Suzuki, K., Imahori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+acetate+kinase+from+Bacillus+stearothermophilus.">Purification and properties of acetate kinase from Bacillus stearothermophilus.</a> J. Biochem. 84, 193-203 (1978).</li></ol>

Çıkar çatışması ilan etti.

Bu testte asetil fosfat algılama hidroksilamin hidroklorür yeterli konsantrasyon ve ferrik klorür çözeltisi konsantrasyonu ve asit bağlıdır. Bu bileşenlerin her iki testin hacmi değişiklik yeniden gözden geçirilmesini gerektirebilir. Enzimatik reaksiyonları burada açıklanan 37 ° yapıldı ° C, 5 dakika 60 ° C hidroksilamin sonlandırma. Bu yüksek sıcaklık asetil hydroxamate kalan asetil fosfat hızlı dönüşüm için izin verecek kritik öneme sahiptir. Absorbans renk geliştirme adım ve ölçü...

<table border="1"><tr><td width="160"> Reaktifi Adı </td><td width="108"> Şirket </td><td width="134"> Katalog numarası </td><td width="103"> Yorumlar </td></tr><tr><td> Asetil fosfat </td><td> Sigma Aldrich </td><td> 01409 </td><td> Lityum tuz (% 97) </td></tr><tr><td> Sodyum fosfat tekbazlı (kurutmak) </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td> Sodyum fosfat dibazik (susuz) </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td> Magnezyum klorür (hekzahidrat) </td><td> ThermoFisher </td><td> M33 </td><td></td></tr><tr><td> Tris Base </td><td> ThermoFisher </td><td> B152 </td><td></td></tr><tr><td> Ferrik klorür (hekzahidrat) </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td> Trikloroasetik asit </td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td> Hidroksilamin hidroklorür </td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td> Adenozin 5&#39;-difosfat sodyum tuzu </td><td> Sigma Aldrich </td><td> A2754 </td><td></td></tr><tr><td> Biomate III Spektrofotometre </td><td> ThermoFisher </td><td> 142982082 </td><td> Standart UV / Vis spektrofotometre </td></tr></table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

Asetat kinaz, asetat ve şeker kinaz-HSP70-aktin (ASKHA) enzim süperailesinin 1-5 bir üyesi, bir substrat olarak ATP kullanan asetil fosfat asetat geri dönüşümlü fosforilasyon için sorumludur. Asetat kinazlar Archaea bir cins bulundu, Bakteriler her yerde, ve aynı zamanda ökaryotlarının 6 mikroplar bulunmaktadır. En iyi karakterize asetat kinaz olduğunu metan üreten archaeon Methanosarcina thermophila 7-14. Sadece asetil fosfat oluşturan yönde ATP i PP yararlanmak ancak bir asetat kinaz Entamoeba histolytica, amip dizanteri etkeni izole edilmiş ve şimdiye kadar sadece bu cinsin 15,16 olmuştur. Asetil fosfat oluşum doğrultusunda, asetat kinaz aktivitesini genellikle ilk Lipmann tarafından açıklanan, hydroxamate tahlil kullanarak ölçülür.17-20, enzimler piruvat kinaz ve laktat dehidrogenaz 21,22 ADP ATP dönüşüm NAD NADH oksidasyonu birleştiğinde olduğu bir birleştiğinde tahlil + veya asetil fosfat ürün reaksiyon sonra inorganik fosfatın serbest ölçen bir test hidroksilamin 23. Etkinlik tam tersi, asetat oluşturan yönü ADP NADP azaltılması + enzimler heksokinaz ve glukoz 6-fosfat dehidrogenaz 24 NADPH, ATP oluşumu birleştirilmesi ile ölçülür . Burada kaplin enzimler gerektirmez asetat oluşumu yönünde asetat kinaz aktivitesinin tespiti için bir yöntem tarif, ama onun yerine asetil fosfat tüketimi doğrudan belirlenmesi üzerine dayanır. Enzimatik reaksiyon sonra, kalan asetil fosfat hydroxamate tayini için spektrofotometrik olarak ölçülmüş olabilir ferrik hydroxamate karmaşık dönüştürülür. Böylece, I. aksineADP ATP üretimine bağımlı bu yönde andard birleştiğinde tahlil, doğrudan bu testi ATP veya PP i üretmek asetat kinazlar için kullanılabilir.

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Asetat kinaz aktivitesinin belirlenmesi için bir yöntem açıklanmıştır. Bu testte, asetat oluşturan yönde asetat kinaz enzim aktivitesi ve kinetiği farklı fosforilasyonunu alıcıları belirlemek için doğrudan bir tepki kullanır. Ayrıca, bu yöntem diğer asetil fosfat ya da asetil-CoA kullanan enzimler assaying için kullanılabilir.

Enzim Asetat Kinaz Asetat oluşturan Faaliyet Doğrudan Algılama

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

direct-detection-acetate-forming-activity-enzyme-acetate

Research

JoVE Journal

Biology

23.0K Görüntüleme.  Clemson University. Asetat kinaz aktivitesinin belirlenmesi için bir yöntem açıklanmıştır. Bu testte, asetat oluşturan yönde asetat kinaz enzim aktivitesi ve kinetiği farklı fosforilasyonunu alıcıları belirlemek için doğrudan bir tepki kullanır. Ayrıca, bu yöntem diğer asetil fosfat ya da asetil-CoA kullanan enzimler assaying için kullanılabilir.

Video: Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Protein Ubikitinasyon Tespiti

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Biyoloji

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

Proteaz Aktivitesi belirleme Proteaz Floresan Algılama Kit kullanın

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Mikotoksin Algılama ELIME (Enzim İmmüno Manyetik Elektrokimyasal Bağlantılı) Yöntem

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Transgenik, üretimi Xenopus laevis Kısıtlama Enzim aracılığı ile Entegrasyon ve Nükleer Nakli

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Nitrik Oksit ve hücreler arası Elektron Paramanyetik Rezonans Spektroskopisi ile süperoksit Radikal Anyon tespiti Spin Tuzakları kullanarak

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Hücre Döngüsü FRET oranlı metrik sayesinde Kinaz ve Fosfataz Aktiviteleri İzleme

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2 Kinaz Aktivitesi Assaying In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

Toprak Mikrobiyal Kalıntı Tayini için Aldononitrile Asetat türevlendirilmiş Glukozamin ve Muramic Asit GC tabanlı Algılama

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Larvaların Faaliyet Ölçümü<em&gt; Drosophila</em&gt; Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

Yetişkin Sivrisinekte Nöropeptitlerin ve Boşaltım Sisteminin Diğer Düzenleyicilerinin Aktivitesinin Incelenmesi

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...