Bu eser R &amp; D Systems, Inc tarafından finanse edilmiştir

1. Örnek Hazırlanması <ol><li> İnsan Fosfo-RTK dizi veri sayfasındaki yönergeleri izleyerek tedavi edilmezse, ligand tedavi veya önleyici ve ligand tedavi hücrelerinden lizatları toplayın. Lizis tamponu bu kit için optimize edilmiştir. Diğer lizis tampon ikame dizisinin nihai performansını etkileyebilir. Proteolitik örnek düşmeyi önlemek için, 10 ug / ml Aprotinin, 10 ug / ml Löpeptin, 10 ug / ml Pepstatin her kullanım için taze her bir hücre lizatı hazırlanması için gerekli lizis tampon hacmi A ilave edin. </li><li> Lizat konsantrasyonu bicinchoninic asit (BCA) deneyi kullanılarak ölçülür. Numune konsantrasyonu ampirik optimum hassasiyet ve düşük arka plan için ayarlanabilir. Lizat 100-300 mikrogram Bir dizi, bir başlangıç ​​noktası olarak tavsiye edilir. </li><li> Numunelerin donma / çözülme döngüleri kaçınılmalıdır ve lizatları uygun depolama optimum performans için gereklidir. Buz üzerinde donmuş lizat örnekleri çözülme. </li></ol><ol><li> Kullanmadan önce oda sıcaklığına kadar tüm kit bileşenleri getir. Işlemleri yaparken kontaminasyonu önlemek için eldiven giyin. </li><li> Kullanılacak 4-Multi-Well çanak her oyuğuna Dizi Tampon 1 2.0 ml. Dizi Tampon 1 blok tampon olarak hizmet vermektedir. </li><li> Düz uçlu cımbız kullanarak, 4-İyi Multi-çanak bir kuyuya koruyucu çarşaf ve yerde arasından kullanılmak üzere her membran kaldırmak. Dizi numarasını yukarıya dönük olmalıdır. </li><li> Dizi Tampon 1 ile temas ettiğinde noktalar mavi boya kaybolur, ancak yakalama antikorlar kendi özel yerlerde tutulur. </li><li> Sallanan bir platform üzerinde 1 saat süreyle inkübe edin. Orient tepsiye böylece her dizi kayalar sonunda onun da içinde biteceğini söyledi. Dizinin yüzeyi tamamen ıslatılmalı ve blok tampon ile kaplanmış olmalıdır. </li><li> Membranlar bloke edilirken, Dizi Tampon 1 ile 1,5 ml nihai hacmi hücresel lizat istenilen miktarda sulandırın. </li> &lt;li&gt; Aspiratif Array 4-Peki Multi-çanak kuyulardan Tampon 1 ve seyreltilmiş lizat çözüm ekleyin. 4-Peki Multi-çanak kapağı yerleştirin. </li><li> Sallanan bir platform üzerinde 2-8 ° C&#39;de bir gece inkübe edin. Optimum hassasiyet gerekli değilse daha kısa inkübasyon süresi kullanılabilir. </li><li> Dikkatlice 1X Yıkama Tamponu 20 ml ile bireysel plastik kaplar içine 4-Peki Multi-çanak ve yerden her membran kaldırmak. 4-Peki Multi-bulaşık yıkama adımları tamamlamak için kullanılan olabilir. Deiyonize veya distile su ile 4-Peki Multi-çanak iyice durulayın ve kurulayın. </li><li> Bir sallanan bir platform çalkalayıcı üzerinde 10 dakika süreyle 1X Wash Buffer ile her bir membran yıkayın. 3 yıkar toplam iki kez tekrarlayın. </li><li> Flakon etiketi üzerindeki seyreltme faktörü kullanarak 2 1X Array Tampon içinde anti-fosfo-tirozin-horseradish peroksidaz (HRP) algılama tampon seyreltin. 4-Multi-Well çanak her oyuğuna pipetle seyreltilmiş 2.0 ml. </li><li> Dikkatlice yıkama kabı her membran kaldırmak.Membran tahliye ve seyreltilmiş anti-fosfo-tirozin-HRP içeren 4-Peki Multi-çanak membran dönmek için aşırı arabellek izin ver. Kapaklı kuyuları kaplayın. </li><li> Bir sallanan platform üzerinde oda sıcaklığında 2 saat süreyle inkübe edin. </li><li> Daha önce anlatıldığı gibi, her dizi yıkayın. </li><li> Kesintisiz kalan adımları tamamlayın. Dikkatlice yıkama kabı her membran kaldırmak. Aşırı arabellek emici kağıda alt kenarı blot ile membran dışarı akmasını sağlayın. Yukarı bakacak kimlik numarası ile bir plastik levha koruyucusu her membran yerleştirin. </li><li> Hazırlanan CHEMI reaktifi pipetleyin 1 ml her bir membran üzerine eşit karıştırın. Membran başına CHEMI Reaktif Karışımı az 1 ml kullanılması eksik membran kapsama neden olabilir. Chemi Reaktifler 1 ve 2 için bazı yüksek yoğunluklu chemiluminescent reaktif ikame reaktif bağlı olarak artan veya azalan arka plan sinyal ya da neden olabilir. </li><li> Dikkatli bir plastik kapaklevha koruyucusu. Yavaşça herhangi bir hava kabarcığı yumuşatmak için ve CHEMI Reaktif Karışımı Her membran her köşesine yayılıyor sağlamak. 1 dakika boyunca inkübe edin. </li><li> Pozisyon kağıt üst ve membranlar içeren plastik levha koruyucusu yanlarında havlu ve dikkatle aşırı CHEMI Reaktif Mix sıkmak. </li><li> Üst plastik levha koruyucusu çıkarın ve dikkatle bir emici laboratuvar kalan CHEMI Reaktif Karışımı kapalı leke membranları üstüne silin yatıyordu. </li><li> Alt plastik levha koruyucusu membranlar bırakarak, plastik wrap yavaşça herhangi bir hava kabarcığı düzeltmek için dikkat çekici membranları kapsamaktadır. Membranlar ve levha koruyucusu tamamen sarılmış böylece levha koruyucusu arka etrafında aşırı plastik wrap sarın. </li><li> Radyoaktif izotop algılama ile kullanılmayan bir otoradyografi film kaseti içinde, yukarı bakacak kimlik numaraları ile membranlar yerleştirin. </li><li> 1-10 dakika boyunca Kodak Biomax Işık film Açığa. Çoklu pozlama recomm vardırsona erdi. Alternatif olarak, görüntüleri, Carestream Health olarak kemilüminesans uyumlu kamera kullanılarak toplanabilir Image Station 4000mm PRO. </li></ol> 3. Veri Analizi <ol><li> Geliştirilmiş film görülen olumlu sinyalleri hızla dizi görüntü üzerinde şeffaf kaplama yerleştirmek ve bu amaç için köşe basılmış referans noktaları çiftleri ile hizalayarak ile tespit edilebilir. Dizi damgalı kimlik numarası sol vardı tarafta konulmalıdır. Kontrolleri ve yakalama antikorların konumu İnsan Fosfo-RTK dizi veri sayfasının Ek listelenir. </li><li> Geliştirilen film Piksel yoğunlukları gibi Epson Perfection V750 PRO gibi bir iletim modu tarayıcı kullanılarak toplanabilir. </li><li> Uygun bir görüntü analiz programı kullanılarak dizinin her nokta piksel yoğunluğu analiz etmek için bir şablon oluşturun. Uyumlu programlar GE&#39;den ImageQuant veya ArrayVision yazılım, Batı Vizyon sof dahilProfiler Diziler, BioRad Miktar biri, Adobe Photoshop, NIH ImageJ ve Protein Basit AlphaView proteom özgü şablonları ile tware. </li><li> Microsoft Excel gibi bir programda manipülasyon için bir elektronik tablo dosyasına sinyal değerlerini aktarın. </li><li> Her RTK temsil yinelenen lekeler çiftinin ortalama sinyal (piksel yoğunluğu) belirleyin. </li><li> Her nokta bir ortalama arka plan sinyali çıkarın. Dizi ya da bir arka plan değeri olarak negatif kontrol noktaları açık bir alan bir sinyal kullanın. </li><li> Numuneler arasındaki RTK fosforilasyon seviyeleri göreceli değişiklik belirlemek için farklı dizilerde karşılık gelen sinyalleri karşılaştırın. </li></ol> 4. Temsilcisi Sonuçlar Bu protokol, insan Fosfo-RTK dizisi RTK fosforilasyonu üzerinde ligand uyarılması ve inhibitörlerinin etkilerini taranması için nasıl kullanılabileceğini göstermektedir. Şekil 1 &#39;de, veri 100 ng ile muamele edilmiş, tedavi edilmemiş olan MDA-MB-453 hücreleri için gösterilir5 dk, veya bilinen ErbB aile inhibitörleri önce HRGβ1 tedavi için 8-11 ile önceden tedavi edilen / ml rhNRG-β1/HRG-β1 (HRG-β1). Inhibitörü deneyler için, hücreler 1 uM HDS 029, 200 nM PD 153035, ya da üç saat boyunca 200 nM SFM PD 158780 ile birlikte inkübe edildi. Her dizinin lisatı 100 ug ile inkübe edildi. Fosforilasyon bir artış ErbB2, ErbB3 için gözlendi ve ErbB4 yakalama noktalar HRG-β1 ile tedavi edilmemiş hücrelerin karşılaştırma MDA-MB-453 hücrelerinin tedavi edilir. Önceki HRG-β1 inkübasyon için her üç ErbB aile selektif inhibitörleri ile hücrelerin tedavi ErbB2 azalmalar, ErbB3, ErbB4 ve fosforilasyon neden olmuştur. Şekil 2, veri RTK FGF, R2 overexpress için bilinen Kato III hücrelerine için gösterilir. Bu deney kümesinde, Kato III hücrelerine önce ile muamele ile 100 ng / ml rhFGF asidik (FGF) ve 1 ug / ml heparin, ya da FGF R 12-15 selektif inhibitörleri ile işlemden geçirilmiş ile muamele edilmiş, tedavi edilmemiş edilerekFGF ve heparin. Inhibitörleri PD173074, PD 161570, PD 166285 ve 1 uM bir konsantrasyonda kullanılmakta ve serumsuz ortam içinde, 3 saat boyunca Kato III hücre ile enkübe edildi. EGF R ve işlenmemiş KATO III hücrelerinde FGF R2 hem kurucu fosforilasyon, FGF R2 fosforilasyon bir artış olsa da FGF tedavisi üzerine gözlenmiştir. PD 173074 ile hücreler inkübe, PD 161570, PD 166285 veya FGF, R2 ve EGF R fosforilasyon azalma ile sonuçlanmıştır. Bu inhibitörler FGF R aile üyelerinin fosforilasyon etkiler bilinmesine rağmen, bu sonuçlar, inhibitör spesifik bir konsantrasyon, bu durumda bu tür EGF R gibi diğer RTKlar, üzerinde olası etkileri izlenmesi için insan fosfo-RTK dizi fayda gösterir. <img alt="Şekil 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/4199/4199fig1highres.jpg" src="/files/ftp_upload/4199/4199fig1.jpg" /> Şekil 1. Recep indüksiyonu ve inhibisyonumeme kanseri hücrelerinde tor tirozin kinaz fosforilasyon. Proteome Profiler&#39;ı İnsan fosfo-RTK dizileri ve karşılık gelen histogram profillerden Dizi görüntüleri gösterilir. MDA-MB-453 hücrelerinin tedavi edilmemiş ya da RTK inhibitörleri NRG-β1/HRG-β1 ile Örnek ile muamele edildi. Dizi MDA-MB-453 hücrelerinde kinaz fosforilasyon üzerine inhibitörlerinin etkilerini gözlemlemek için kullanıldı. <a href="http://www.jove.com/files/ftp_upload/4199/4199fig1large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> . <img alt="Şekil 2," fo:content-width="4.5in" fo:src="/files/ftp_upload/4199/4199fig2highres.jpg" src="/files/ftp_upload/4199/4199fig2.jpg" /> Şekil 2. Bir Proteome Profiler İnsan fosfo-RTK dizisi ve karşılık gelen histogram profillerden indüksiyonu ve mide kanseri hücrelerinde reseptör tirozin kinaz fosforilasyon inhibisyonu. Görüntüler dizisi gösterilir. Kato III hücreler unt vardıreated veya FGF asidik ve heparin ile tedavi takip RTK inhibitörleri ile tedavi. <a href="http://www.jove.com/files/ftp_upload/4199/4199fig2large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> . Referanslar <ol><li> Henriksson&#39;a ML, Edin, S., Dahlin, AM, Oldenborg, PA, Oberg, A., Van Guelpen, B., Rutegard, J., Stenling, R., ve Palmqvist, R. Kolorektal kanser hücrelerinin aktive edilen bitişik fibroblastlar FGF1/FGFR3 sinyalizasyon ve artan invazyonu. Am. J. Pathol. 178, 1387-1394 (2011). </li><li> Morishita, A., Gong, J., Nomura, T., Yoshida, H., Izuishi, K., Suzuki, Y., Kushida, Y., Haba, R., D&#39;Armiento, J., ve Masaki, T. protein dizisinin kullanılması, insan kolon kanseri tedavisi için, hedeflenebilir reseptör tirozin kinazlar tanımlamak için. Int. J. Oncol. 37, 829-835 (2010). </li><li> Agaram, NP, Laquaglia, MP, Üstün, B., Guo, T. Wong, GC, Socci, ND, Maki, RG,Pediatrik gastrointestinal stromal tümörlerin DeMatteo, RP, Besmer, P., &amp; Antonescu, CR moleküler karakterizasyonu. Clin. Kanser Araş. 14, 3204-3215 (2008). </li><li> Stommel, JM, KimmelKimmelman, AC, Ying, H., Nabioullin, R., Ponugoti, AH, Wiedemeyer, R., Stegh, AH, Bradner, JE, Ligon, KL, Brennan, C., Chin, L., &amp; DePinho, reseptör tirozin kinaz RA Coactivation hedefe yönelik tedaviler, tümör hücrelerinin tepkisini etkiler. Bilim. 318, 287-290 (2007). </li><li> Agarwal, S., Zerillo, C., Kolmakova, J., Christensen, JG, Harris, LN, Rimm, DL, DiGiovanna, MP, ve Stern, bir direnç ile yapısal aktive hepatosit büyüme faktörü reseptörünün DF Derneği (Met) küçük olmayan hücreli akciğer kanseri hücrelerinde çift EGFR/Her2 inhibitörü. Br. J. Kanser. 100, 941-949 (2009). </li><li> Dewaele, BM Floris, G., Finalet-Ferreiro, J., Fletcher, CD, Coindre, JM, Guillou, L., Hogendoorn, PC, Wozniak, A., Vanspauwen, V., Schoffski, P., MARYNEN, P., Vandenbergh, P., Sciot, R., ve Debiec-Rychter, M. Co-aktif PDGFRA ve EGFR intimal sarkom potansiyel terapötik hedeflerdir. Kanser Araş. 70, 7304-7314 (2010). </li><li> Yoon, YK, Kim, HP, Han, GB, Hur, HS Oh, DY, Im, SA, Bang, YJ, &amp; Kim, EGFR ve MEK1 / 2 inhibitörü TY Kombinasyon baskılayarak EGFR/HER3-dependent tarafından sinerjistik etki gösterir insan mide kanseri hücreleri AKT aktivasyonu. Mol. Kanser Ther. 8, 2526-2536 (2009). </li><li> Fry, DW, Nelson, JM, Slintak, V., Keller, PR, Rewcastle, GW, Denny, WA, Zhou, H., ve Köprüler, 4 AJ Biyokimyasal ve antiproliferatif özellikleri - [Ar (alk) ilamino] piridopirimidinler, güçlü ve spesifik bir epidermal büyüme faktörü reseptör tirozin kinaz inhibitörlerinin, yeni bir kimyasal sınıf. Biochem. J. Pharmacol. 54, 877-887 (1997). </li><li> Bos,M., Mendelsohn, J. Kim, YM, Albanell, J. Fry, DW, &amp; Baselga, J. PD153035, bir tirozin kinaz inhibitörü, epidermal büyüme faktörü reseptör aktivasyonu engeller ve bir reseptör kanser hücrelerinin büyümesini engeller sayısı- bağımlı bir tarzda, J. Clin.. Cancer Res. 3, 2099-2106 (1997). </li><li> Stoll, SW, Kansra, S., Peshick, S., Fry, DW, Leopold, WR, Wiesen, JF, Sibilia, M., Zhang, T., Werb, Z., Derynck, R., Wagner, EF, &amp; Elder, normal ve malign keratinositler kıyasla deride ErbB reseptör tirozin kinaz JT Diferansiyel kullanımı ve yerelleştirme. Neoplazi. 3, 339-350 (2001). </li><li> Klutchko, SR, Zhou, H., Winters, RT, Tran, TP, Köprüler, AJ, Althau, IW, Amato, DM, Elliott, WL, Meade, Fry MA, Roberts, BJ, DW, Gonzales, AJ, Harvey , PJ, Nelson, JM, Sherwood, V., Han, HK, Pace, G., pencere Classic, JB, Denny, WA, ve Showalter, HD Tirozin kinaz inhibitörleri. 19. 6-alktirozin kinaz reseptörleri ailesinin bir ErbB tersinmez inhibitörleri olarak 4-anilinoquinazolines ve 4-anilinopyrido [3,4-d] pirimidin nyamides. J. Med. Chem. 49, 1475-1485 (2006). </li><li> Kunii, K., Davis, L., Gorenstein, J., Hatch, H., Yashiro, M., Di Bacco, A., Elbi, C., ve Lutterbach, B. FGFR2 amplifiye mide kanseri hücre hattı FGFR2 gerektirir ve Erbb3 büyüme ve hayatta kalmak için sinyalizasyon. Kanser Araş. 68, 2340-2348 (2008). </li><li> Zhou, W., Hur, W. McDermott, U., Dutt, A., Xian, W., Ficarro, SB, Zhang, J., Sharma, SV, Brugge, J., Meyerson, M., Settleman, J . &amp; Gray, NS kovalent FGFR inhibitörleri oluşturmak için bir yapı güdümlü bir yaklaşım. Chem. Biol. 17, 285-295 (2010). </li><li> PD 161570 tarafından FGF-1 reseptör tirozin kinaz aktivitesi Batley, BL, Doherty, AM, Hamby, JM, Lu, GH, Keller, P., Dahring, TK, Hwang, O., Crickard, K., &amp; Panek RL inhibisyonu yeni bir protein-Tirosin kinaz inhibitörüdür. Life Sci. 62. 143-150 (1998). </li><li> Panek, RL, Lu GH, Klutchko, SR, Batley, BL, Darhing, TK, Hamby, JM, Hallak, H., Doherty, AM, ve Keiser, PD 166285 in vitro farmakolojik karakterizasyonu JA, yeni nanomolar güçlü ve geniş aktif protein tirozin kinaz inhibitörü. J. Pharmacol. Tec. Ther. 283, 1433-1444 (1997). </li></ol>

İnsan Fosfo-RTK gibi RTK fosforilasyon tarama değişiklikler için Batı immünopresipitasyon (IP) gibi geleneksel yöntemlere ekonomik ve hızlı bir alternatiftir. Bu yöntemi kullanarak, 42 ​​fosfo-RTKlar göreli MDA-MB-453 veya Kato III hücre lizat tek bir örnek kullanılarak eş zamanlı tarandı. Buna ek olarak, bu dizi deney bu yöntem çok daha fazla zaman etkili birden fazla IP-Western değerlendirme yerine yapma, 2.5 saat eller zaman gereklidir. Bir kemilüminesans algılama yöntemi kullanılarak, genellikle Batı veri toplamak için kullanılan ötesinde hiçbir özel ekipman gerekli oldu. Diziler hassas olan ve ligand ve inhibitörü ile meydana fosforilasyon değişiklikleri karşılaştırmak için kullanılabilir. Bu yöntem aynı zamanda off-hedef RTKlar için önleyici seçicilik değerlendirilmesine olanak tanımaktadır. Pozitif isabet ayrıca, bu tür bir ELISA gibi bir miktar tayini kullanılarak değerlendirilebilir. Etkili değişiklikler için ekrana diziler istihdam etmekfosforilasyonu, bazı temel deneysel kurallara uyulmalıdır. İlk deneyler uygun kontrol numuneleri içermelidir. Örneğin, bu deneylerde MDA-MB-453 veya Kato III lizatları üzerine inhibitörlerinin etkisi aynı gün hazırlanan lizat örneklerinde ölçüldü. Aynı deney içinde toplanan Sadece dizi veri numune işleme, kuluçka süresi, pipet tekniği ya da yıkama tekniği nedeniyle sinyal değişimleri en aza indirmek için analiz edilmelidir. Yakalama antikorlar paralel yakınlıkları olması seçilmemiştir beri aynı zarı iki analit arasındaki karşılaştırma sinyal intensitesi, tavsiye edilmez.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td><a href="http://www.rndsystems.com/product_results.aspx?k=ary001" target="_blank">Human Phospho-RTK Array Kit</a></td>
			<td>R&amp;D Sytems</td>
			<td>ARY001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Tocris</td>
			<td>1167</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>Tocris</td>
			<td>1190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MDA-MB-453 cells</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kato III cells</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td><a href="http://www.rndsystems.com/product_results.aspx?k=396-hb" target="_blank">Recombinant Human NRG1-beta 1/HRG1-beta 1 EGF Domain</a></td>
			<td>R&amp;D Systems</td>
			<td>396-HB-050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td><a href="http://www.rndsystems.com/product_results.aspx?k=232-fa" target="_blank">Recombinant Human FGF acidic (aa 16-155)</a></td>
			<td>R&amp;D Systems</td>
			<td>232-FA-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HDS 029</td>
			<td>Tocris</td>
			<td>2646</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 153035</td>
			<td>Tocris</td>
			<td>1037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 158780</td>
			<td>Tocris</td>
			<td>2615</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 173074</td>
			<td>Tocris</td>
			<td>3044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 161570</td>
			<td>Tocris</td>
			<td>3724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 166285</td>
			<td>Tocris</td>
			<td>3785</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BCA assay</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate-Buffer Saline (PBS)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettes and pipette tips</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Deionized or distilled water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flat-tipped tweezers</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rocking platform shaker</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plastic containers with the capacity to hold 50 ml</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>For washing arrays</td>
		</tr>
		<tr>
			<td>Plastic transparent sheet protector</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plastic wrap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Absorbent lab wipes</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Autoradiography cassette</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Film developer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kodak BioMax Light Film</td>
			<td>Carestream Health</td>
			<td>178 8207</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Film developer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Image Station 4000MM PRO</td>
			<td>Carestream Health</td>
			<td>&nbsp;</td>
			<td>Chemiluminescence imager compatible with collection of western data may be used instead of film and a film developer.</td>
		</tr>
		<tr>
			<td>Epson Perfection Scanner</td>
			<td>Epson</td>
			<td>V750 PRO</td>
			<td>Flatbed scanner with transparency adaptor capable of transmission mode</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>GE,WesternVision, BioRad, Adobe, NIH, Protein Simple, etc</td>
			<td>&nbsp;</td>
			<td>ImageQuant, ArrayVision, Western Vision, Quantity One, Photoshop, ImageJ, AlphaView, or other compatible program</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Computer capable of running image analysis software and Microsoft Excel.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

profiling changes in receptor tyrosine kinase phosphorylation using antibody arrays - advertisement

Reseptör tirozin kinaz (RTK) ekspresyonu ve fosforilasyon düzensizliği sık kanserli hücrelerin gelişimini ve metastazı ile ilişkili. 1-3 önemli çaba belirli RTKlar hedeflemek ve hastalık durumları ile ilişkili anormal sinyal yolları kesmek için gelişmekte inhibitörleri odaklanmıştır . Bu çalışmanın 4-7 amacı RTK fosforilasyonuna inhibitörleri, belirli bir sayıda etkilerini ölçmektir. Bu çalışmada, tek bir örnek aynı anda çok sayıda RTKlar için fosforilasyonu artar veya azalır izlemek için bir membran tabanlı sandviç kitidir İnsan Fosfo-RTK dizisi kullanılır. Bu denemede, her ikisi de fosforile ve fosforlanmamış RTKlar bir lizat numune içinde mevcut bir nitroselüloz zar, bir mikroskop slaydı büyüklüğü karşısında iki kopya halinde basılmış farklı antikorları tarafından yakalanır. Yıkamadan sonra, diziler, anti-fosfo-tirozin-yaban turpu peroksidaz (HRP) ile kuluçkaya bırakıldıdizi yakalanan fosforile RTKlar ile hangi sandviç. Bir ikinci yıkama adımından sonra, diziler chemiluminescent reaktifleri ile inkübe edilir. Her dizi noktada üretilen sinyal her yakalama antikorla bağlanır fosfo-protein miktarı ile doğru orantılıdır. Bu deneylerde, diziler MDA-MB-453 göğüs kanseri ya da Kato III mide kanseri hücreleri, hem de ErbB karşı selektif inhibitörleri ile hücrelerin tedavisi ile ilişkili fosforilasyon azalmasına karakterize etmek için her iki ligand uyarımını takiben fosforilasyon artışı ölçmek için kullanılmıştır veya FGF R aile üyeleri.

Proteom Profiler antikor diziler sayıda immünopresipitasyon (IP) Westernler yapmadan reseptör tirozin kinaz (RTK) fosforilasyon değişiklikler için ekran için uygun ve düşük maliyetli bir yoldur. ARY001 İnsan RTK dizi kemilüminesans algılama kullanarak tek bir örnek birden RTKlar niteliksel olarak ölçülmesini sağlar.

Antikor Diziler kullanarak Tirozin Kinaz Fosforilasyon profilleme değişiklikler - Reklam

The dysregulation of receptor tyrosine kinase (RTK) expression and phosphorylation is frequently associated with the development and metastatic spread of ...

profiling-changes-receptor-tyrosine-kinase-phosphorylation-using

Without Section

Profiling Changes in Receptor Tyrosine Kinase Phosphorylation using Antibody Arrays - ADVERTISEMENT

11.5K Görüntüleme.  R&D Systems, Inc.. Proteom Profiler antikor diziler sayıda immünopresipitasyon (IP) Westernler yapmadan reseptör tirozin kinaz (RTK) fosforilasyon değişiklikler için ekran için uygun ve düşük maliyetli bir yoldur. ARY001 İnsan RTK dizi kemilüminesans algılama kullanarak tek bir örnek birden RTKlar niteliksel olarak ölçülmesini sağlar.

Video: Antikor Diziler kullanarak Tirozin Kinaz Fosforilasyon profilleme değişiklikler - Reklam

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state Nanopores ile izlenmesi Protein Adsorpsiyon

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Research

JoVE Journal

Biyomühendislik

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Klinik Antikor Örnekleri fagositik Aktivitesi Belirlenmesi

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

İmmünoloji ve Enfeksiyon

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

T Hücreleri İçin Kantitatif Yüksek throughput Tek hücreli Sitotoksisite Testi

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Içinde Synaptic florasan Otomatik Sayısallaştırma<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Nörobilim

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Antikor Profil Lusiferaz Immunoprecipitation Sistemleri (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Biyoloji

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western Blot: Numune Hazırlama Algılama

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood 1,2,3. Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) 1,2 and precocious puberty 4. Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR.
Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well.
The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies 1,4,5,6,7,8. As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation 4 as well as to alter the rate of degradation of KISS1R 9. Interestingly, our studies indicate that KISS1R is degraded by the proteasome, as opposed to the classic lysosomal degradation described for most G protein-coupled receptors 9. In the example presented here, degradation of the KISS1R is investigated in Human Embryonic Kidney Cells (HEK-293) transiently expressing Myc-tagged KISS1R (MycKISS1R) and treated with proteasome or lysosome inhibitors. Cell lysates are immunoprecipitated using an agarose-conjugated anti-myc antibody followed by western blot analysis. Detection and quantification of MycKISS1R on blots is performed using the LI-COR Odyssey Infrared System. This approach may be useful in the study of the degradation of other proteins of interest as well.

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

Üreme Bozuklukları ile İnsanlardaki Belirlenen GC-zengin KISS1 Reseptör sırası genetik mutasyonlar Mutagenez ve Analiz

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence 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 ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Multiplexed kemostat Diziler Tasarımı ve Kullanımı

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...