Bu eser bir Ellison/AFAR doktora sonrası bursu SJK için eskime araştırma programı tarafından desteklenen ve Glenn Vakfı Ödülü ve NIH verir PC için (1P01AG034906, 1R01GM 090311, 1R01ES 020812). Tüm yazarlar filmde görünür.

1. peptid tedavi hücrelere <ol><li>iki milyon SH-SY5Y plaka veya HEK293 hücrelere 10 cm (2 x 10 6) yemek ve onlar için 2 gün</li> <li>(isteğe bağlı) ertesi gün, Dulbecco serum içermeyen hücrelerle yıkama &#39; s modifiye Orta (DMEM) medya bir kez kartal ve ücretsiz DMEM gecede serum boş ortam içinde yapılması gereken tedavi ihtiyacı varsa serum ile kuluçkaya.</li>
	<li>3 günde S14G-humanin peptidler µm filtre, distile su ve 1 mM hisse senedi çözüm olarak sulandırmak 0,2 içinde erimesi. 
	Not: Peptidler peptid (örneğin, genel ücret) sıra özellikleri tarafından belirlenen ve peptidler ticari olarak kullanılabilir durumdaysa üreticisi tarafından sağlanan uygun çözücü içinde çözünmüş. Örneğin, S14G-humanin, SHLP2 ve SHLP6 ve MOTS-c suda çözülür. Aliquot peptidler döngüleri erime ve donma önlemek için bir uygun birimi (örneğin, 50 μL) içinde. Bir kez çözdürülen, bunları tekrar kullanmayın.</li>
	<li>Aliquot Önceden ısıtılmış serum içeren veya bir 50 ml konik tüp ve bir çalışma konsantrasyonu (örneğin, 1 µM, 10 µM) yapmak için oda sıcaklığında 1 mM hisse senedi çözüm eklemek serum-Ücretsiz medya.</li>
	<li>Yerine medya ile 6 mL peptid eriyik--dan adım 1.4. ve hücreleri uygun süreyi için kuluçkaya. 
	Not: kuluçka süresi ERK etkinleştiren, koşula bağlı olarak seçin.</li>
</ol> 2. Sitozol ve çekirdeği için hücre altı ayırma <ol><li>buz gibi PBS 10 mL iki kez hücrelerle yıkama, hücreleri 5 mL buz gibi PBS de plaka kapalı kazımak ve hücre süspansiyon 15 mL konik tüp içine transfer.</li>
	<li>Hücreleri, 500 x g 5 min için saat 4'te santrifüj kapasitesi ° C.</li>
	<li>PBS Aspire edin ve Pelet buz gibi ayırma arabellek 200 µL içinde yeniden askıya alma (10 mM HEPES pH 7,6, 3 mM MgCl 2, 10 mM KCl, %5 (v/v) gliserol, % 1 iyonik olmayan yüzey aktif (C 13 H 22 O (C 2 H 4 O) = n), proteaz/fosfataz inhibitörleri). 
	Not: protein fosforilasyon durumları tutmak için fosfataz inhibitörü olarak iyi proteaz inhibitörü taze eklenmelidir ve örnekleri buza hiç tutulmalıdır zaman. Donma ve erime yinelenen örnekleri döngüleri kaçınılmalıdır. Onları-80 buz gibi önce az miktarda (örneğin 50 μg) içine aliquot örnekleri ° C.</li>
	<li>15 dakika buzda yeniden askıya alınmış Pelet kuluçkaya ve 5 min için 250 x g, saat 4'te santrifüj kapasitesi ° C.</li>
	<li>Süpernatant sitoplazmik kesir olarak toplamak ve Pelet nükleer kesir için devam.</li>
	<li>Hücresel enkaz ve 18.000 x g 4 ° C'de 10 dakika için de diğer kirleri çıkarmak ve sonra süpernatant yeni bir microcentrifuge tüp içine aktarmak için süpernatant santrifüj kapasitesi. Sitozol kesir bu.</li>
	<li>Pelet 200 μL buz gibi yıkama arabellekte resuspend (10 mM HEPES pH 7,6, 1.5 mM MgCl 2, 420 mM NaCl, %25 (v/v) gliserol, 0.2 mM EDTA, proteaz/fosfataz inhibitörleri =) ve 4 ° C'de 5 min için 250 x g, santrifüj</li>
	<li>Süpernatant kaldırmak ve Pelet 100 μL buz gibi nükleer ayıklama arabellekte resuspend (20 mM HEPES pH 7,6, 1.5 mM MgCl 2, 420 mM NaCl, %25 (v/v) gliserol, 0.2 mM EDTA, proteaz/fosfataz inhibitörleri =) ve 10 kez solüsyon içeren temizleyicide (5 üzerinde 10 s s Çekil, % 30 amp.) buz üzerinde</li>
	<li>Santrifüj 18.000 x g 4 ° C'de 10 dk de</li>
	<li>Yeni bir microcentrifuge tüp süpernatant aktarın. Nükleer kesir bu.</li>
	<li>Miktarını BCA tahlil kullanarak protein miktarı</li></ol>
 3. ham mitokondrial kesir <ol><li>buz gibi PBS 10 mL ile her 10 cm çanak hücrelerde yıkama, 5 mL buz gibi PBS ekleyin ve bir hücre kazıyıcı kullanarak hücreleri ayırmak. 
	Not: Western Blot mitokondri, iyi bir verim almak için üç 10 cm yemekleri hücre kullanın.</li>
	<li>Hücre süspansiyon 15 mL konik tüp aktarmak ve her üç 10 cm yemeklerini bir 15 ml konik tüp içine birleştirmek.</li>
	<li>Hücreleri, 10 min 600 x g 4'te santrifüj kapasitesi ° C.</li>
	<li>PBS Aspire edin ve Pelet 1 mL buz gibi mitokondri yalıtım arabellek yeniden askıya alma (10 mM Tris-BEZLERİ, 1 mM EGTA/Tris, 200 mM Sükroz, ayarlamak için pH 7.4 =).</li>
	<li>Buz üzerinde politetrafloroetilen kaplamalı havaneli ile 25 vuruş 2 mL homogenizer hücrelerle homojenize 
	Not: Bu mitokondri bütünlüğünü korumak ve mitokondrial kesir verimi en üst düzeye çıkarmak için kritik bir adımdır. Vuruş sayısı her hücre türü için optimize edilmelidir. Yordamına başlamadan önce homogenizer precool.</li>
	<li>Homogenate bir microcentrifuge tüp aktarmak ve vasıl 600 x g 4 ° C'de çekirdeği ve kesilen hücreleri kaldırmak için 10 dk santrifüj kapasitesi.</li>
	<li>Süpernatant toplamak, yeni bir microcentrifuge tüp aktarmak ve 7000 x g 10 min için de 4'te santrifüj kapasitesi ° C. 
	Not: Pelet gevşemiş, süpernatant özenle toplamak ve Pelet bozmamaya çalışın.</li>
	<li>Süpernatant kaldırmak, pelet 200 μL buz gibi mitokondri yalıtım arabelleği ile yeniden askıya alma ve çözüm için yeni bir microcentrifuge tüp aktarın. 7000 x g 10 dk de tüp 4'te santrifüj kapasitesi ° C.</li>
	<li>Pelet yıkamak için yineleme adım 3.8. Bu adımda yeni bir microcentrifuge tüp süpernatant aktarmak gerekli değildir.</li>
	<li>Süpernatant yıkanmış Pelet kaldır ve mitokondri ile 50 μL RIPA arabelleği içeren Pelet yeniden askıya alma.</li>
	<li>Incubate 10 dakika süreyle buzda süspansiyon</li>
	<li>Süspansiyon 16.000 x g 15dk için de 4'te santrifüj kapasitesi ° C.</li>
	<li>Yeni bir microcentrifuge tüp süpernatant aktarın. Mitokondrial kesir bu.</li>
	<li>BCA tahlil kullanarak protein miktarı ölçmek.</li>
</ol> 4. Western Blot fosfo-spesifik proteinler için <ol><li>bir SDS-polyacrylamide Jel Elektroforez (8-%16 erken jel) gerçekleştirmek ve protein bir PVDF membran 4 ' e transfer.</li>
	<li>Kuluçkaya membran TBST %5 BSA ile (% 0,1 Polysorbate 20) arka plan belirsiz bağlayıcı siteleri engellemek için oda sıcaklığında 30 dk için. 
	Not: fosforile protein tespiti için membran BSA değil süt ile engelleyin. Süt içeren kazein, yüksek nonspesifik sinyal sonuçları bir bol miktarda Fosoprotein.</li>
	<li>Membran 4 ° C'de birincil antikoru (anti-fosfo-ERK1/2) ile bir gecede kuluçkaya.</li>
	<li>Ertesi gün, membran TBST ile yıkayın (% 0,1 polysorbate 20) üç kere oda sıcaklığında 5 min için.</li>
	<li>Membran ile ikincil antikoru (Anti-tavşan HRP) oda sıcaklığında 1 h için kuluçkaya.</li>
	<li>Membran TBST ile yıkayın (% 0,1 polysorbate 20) üç kere oda sıcaklığında 5 min için.</li>
	<li>Membran ECL çözüm ile kuluçkaya ve Image analyzer membran görüntü.</li>
	<li>Şerit arabellek 15 dakika oda sıcaklığında ve yıkama membran ile TBST üç kez 5 dakika süreyle sıyırma ile membran</li>
	<li>4.2-4.7 kullanarak anti-toplam ERK1/2 antikor arasındaki adımları yineleyin.</li>
	<li>Her kesir saflığı kontrol etmek için SDS-sayfa çalıştırmalarına ve işaretleri her bölmesi (örneğin, Okan B1 çekirdeği, sitozol için GAPDH ve TOM20 mitokondri için) için tanıma antikorları kullanarak immunoblots.</li>
</ol>

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Bir hissedar ve Yen CohBar, Inc. bir danışman olarak hizmet vermiştir CohBar, Inc Kelvin danışmanlığını Pinchas Cohen olduğunu

Burada, biz iki farklı hücre tiplerinde humanin peptid-aracılı ERK1/2 Etkinleştirme oluşuyor ve aktif ERK1/2 hücre altı lokalizasyonu (örneğin, peptid, zaman nokta ve hücre doz koşullara bağlı olarak farklı olabilir gösterdi yazın). O humanin göstermiştir-sinyal içinde iki hücre hatları gibi farklı dozda kullanımının humanin gereksinimini farklılıkları açıklayabilir iki farklı reseptörleri22,23, sinyalleri. Bu fizyolojik ...

Ortaya çıkan çalışmalar mitokondrial kaynaklı peptidler (MDP'ler) cytoprotection ve metabolizma1,2,3' te önemli rol oynarlar göstermektedir. Sinyal iletim yolu MDP'ler huzurunda anlama bize hangi çeşitli işlevleri MDP'ler modüle mekanizması görmemizi sağlar. İlk tanımlanan MDP, humanin, hücre dışı sinyal düzenlenir kinaz 1/2 (ERK1/2) artırmak için gösterilmiştir fosforilasyon, reseptör4,5bağlama aracılığıyla. Ancak, ERK1/2 aktivasyon aşağı akım etkisi hala underexplored.
ERK1/2 basamaklı hücresel süreçler yayılması, hücre göç, hücresel metabolizma, hayatta kalma ve Apoptozis6,7,8de dahil olmak üzere çeşitli önemli bir aracı olarak hizmet vermektedir. Tüm alacak için ayrı bu hücresel süreçler, aktivite ve ERK1/2 hücre altı yerelleştirme sıkıca fosfatazlar ve İskele proteinler9,10tarafından düzenlenmektedir. Translasyon modifikasyon ek olarak, dinamik ERK1/2 / mekik onun sinyal işlevi, aktivite ve özgüllük11,12düzenlemektedir. ERK1/2 öncelikle sitozol13' te lokalize edilmiştir. Ankraj ve İskele proteinler bir dizi yardımcı organelleri, yüzey veya dağınık sitoplazma13' te hücre iskeleti elemanları ERK1/2 korumak. Stimülasyon, üzerine ERK1/2 fosforile ve onun ankraj proteinler ERK1/2 translocation çekirdeği, mitokondri, Golgi ve organellerin14, de dahil olmak üzere diğer hücre altı bölmeleri için sağlayan ayrışmış olur 15 , 16.
Humanin ERK1/2 yolu sinyal etkinleştirmek için bilinen rağmen ERK1/2 aktivasyonu sadece toplam hücresini lysates görülmektedir. ERK1/2 hücre altı yerelleştirme aşağı akım etkisi, hem hücre altı yerelleştirme analizi ve toplam düzeyde önemli bir rol oynar bu yana daha önce açıklandığı gibi fosforile ERK1/2 kapsamlı bir anlayış sağlamak için gerekli humanin kaynaklı ERK1/2 harekete geçirmek ve aşağı akım hedefleri aktivasyonu.
Aktif ERK1/2 hedef organelleri anlamak için hücre altı ayırma fosforile ERK1/2 için western Blot tarafından takip gerçekleştirildi. Standart laboratuar ekipmanları ve Kimyasalları kullanır gibi bu yöntem kolayca uygulanabilir. İzole hücre altı bölmeleri delikanlı yorumlanması için sonuçlar sağlayan yüksek saflık vardır. Immunostaining ERK1/2 benzer sonuçlara neden olabilir. Ancak, belirli hücre altı bölmeleri nispeten zor görselleştirmek ve özel fiksasyon ve permeabilization yöntemleri gerektirir. Hücre altı bölmeleri ERK1/2 düzeyleri farklılık ve bu değişim tüm cep telefonu lysates bakıldığında yanlış pozitif ve yanlış olumsuz sinyaller neden olabilir. Bu nedenle, bir immunoblot izole hücre altı bölmeleri kullanarak bize ERK1/2 yerelleştirme daha iyi bir anlayış verir.
Yöntem çok yönlülük değişiklikler diğer MDP'ler veya translocation ile STAT3 başka sinyal moleküllerin de dahil olmak üzere diğer uyarıcı etkilerini araştırmak için iletişim kuralı sağlar. Son zamanlarda keşfedilen küçük humanin benzeri peptidler (yelken) nerede humanin kodlanır ve onlar-si olmak benzer ancak farklı özellikleri HN17' ye göre 16S rRNA bölgesinden kodlanır. Örneğin, 5 dk. ERK1/2 İnceleme yanıt-e doğru farklı peptidler hücre altı yerelleştirme içinde ERK1/2 humanin etkinleştirir, ancak 8 h bize Bu peptidler Biyoloji daha iyi bir anlayış verir sonra SHLP2 ve SHLP3 ERK1/2 etkinleştirin. Kanıt ortaya çıkan sinyal molekülleri, hücre altı yerelleştirme aşağı akım etkileri önemli bir rol oynar gösterdi. Örneğin, STAT3 geleneksel olarak öncelikle resting hücrelerdeki sitozol yerelleştirilecek bilinmektedir ve sonra gen ekspresyonu sitokinler18yanıt olarak etkinleştirmek için çekirdek içine translocates. STAT3 Ayrıca mitokondri için translocates ve TCA döngüsü ve ATP üretimi19düzenlemektedir. Autophagy Yönetmeliği ile ilgili çeşitli yolları20autophagy STAT3 farklı hücre altı yerelleştirme modüle. Örneğin, nükleer STAT3 transcriptionally autophagy ile ilgili genleri düzenler ve bir autophagy modülatör davranır. Sitoplazmik STAT3 moleküller sinyal autophagy ile etkileşerek autophagy yapısal engeller. Mitokondrial STAT3 engeller ve oksidatif stres indüklenen autophagy baskılayarak mitophagy engeller. Bu nedenle, bu hücre altı yuvası ayırma yöntemi diğer sinyal molekülleri yanı sıra ERK1/2 rolünün anlaşılması için önemlidir.

<table>
	<tbody>
		<tr>
			<td>p44/42 MAPK (ERK1/2)</td>
			<td>Cell signaling</td>
			<td>9102</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td>
			<td>Cell signaling</td>
			<td>4370</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>Lamin B1</td>
			<td>Cell signaling</td>
			<td>12586</td>
			<td>Nuclear Marker, Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>Cell signaling</td>
			<td>5174</td>
			<td>Cytoplasmic marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>Tom20</td>
			<td>Santa cruz</td>
			<td>SC-17764</td>
			<td>Mitochondria marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>anti-Rabbit-HRP conjugated</td>
			<td>Cell signaling</td>
			<td>7074</td>
			<td>Dilution 1:30,000</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Culture Dish</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>12556002</td>
			<td></td>
		</tr>
		<tr>
			<td>HNG peptide</td>
			<td>Genescript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25mm sylinge filter</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>09-719A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>3609</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HCL</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>FALCON</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt&#8482; Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td>
			<td>Thomas Scientific</td>
			<td>3432S90</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td>
			<td>BIO RAD</td>
			<td>4561104</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Module</td>
			<td>BIO RAD</td>
			<td>1658030</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>BIO RAD</td>
			<td>1704150</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td>
			<td>BIO RAD</td>
			<td>1704272</td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Blotting Substrates</td>
			<td>BIO RAD</td>
			<td>1705060</td>
			<td></td>
		</tr>
		<tr>
			<td>Restore Western blot stripping buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>21059</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator, Medel: FB120</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>695320-07-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

Mitokondrial kaynaklı peptidler (MDP'ler) küçük açık okuma çerçevesi mitokondri genom kopyası bilinen diğer genler içinde tarafından kodlanmış peptidler yeni bir sınıf vardır. MDP'ler çok çeşitli nöronların apoptosis korumak, metabolik işaretçileri artırma ve kemoterapi hücrelerin korunması gibi biyolojik etkileri var. Humanin ilk MDP keşfedilmeyi ve en çok çalışılan peptid MDP aile arasında. Membran reseptörleri ve humanin aşağı akım sinyal yollar dikkatle karakterize. MOTS-c ve SHLP1-6 gibi ek MDP'ler daha Geçenlerde bulunduktan ve sinyal mekanizmaları henüz aydınlatılmamıştır zorunda. Burada Bu peptidler işlevini belirlemek için bir hücre kültür dayalı yöntem açıklanmaktadır. Özellikle, hücre ayırma teknikleri, western Blot birlikte harekete geçirmek kantitatif tayin ve translocation önemli sinyal molekülünün için izin verir. Hücre ayırma başka yöntemler olmakla birlikte, burada açıklanan bir kolay ve basit yöntemdir. Bu yöntemler Bu peptidler ve diğer terapötik ajanlar etki mekanizması daha fazla aydınlatmak için kullanılabilir.

Burada sunulan işlemi uygulayarak, biz 1 mikron ve 100 mikron HEK293 ve SH-SY5Y hücrelerle tedavi S14G-humanin, bir güçlü humanin analog21, sırasıyla, içinde tamamlamak medya belirtilen süreler için (Resim 1A ve şekil 1 B). sonra ERK1/2 Toplam ve fosforile şeklinde Thr202/Tyr204 toplam proteini özleri itibariyle incelenmiştir. S14G-huma...

Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Bu iletişim kuralı, mitokondrial kaynaklı peptidler içeren hücreleri uyarmak ve sinyal cascade ve yerelleştirme fosfo-proteinlerin değerlendirmek açıklar.

Mitokondrial kaynaklı peptid tedavi üzerine ERK harekete geçirmek için hücre altı ayırma

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

subcellular-fractionation-for-erk-activation-upon-mitochondrial

Research

JoVE Journal

Biology

7.8K Görüntüleme.  University of Southern California. Bu iletişim kuralı, mitokondrial kaynaklı peptidler içeren hücreleri uyarmak ve sinyal cascade ve yerelleştirme fosfo-proteinlerin değerlendirmek açıklar.

Video: Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Xenopus laevis Yumurta hazırlanması ve Fraksiyonu ayıklar.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Biyoloji

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

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

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

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

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

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Yapışkan ve Sigara yapışık Memeli Hücre yerinde Subselüler Fraksiyonu

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

RhoC GTPaz Etkinleştirme Testi

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

İyon Kanalları ve Fonksiyonel Analiz Mutagenez Heterologously Memeli Hücreleri cinsinden ifade edilir

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Mukayeseli Proteomik Analizi Metabolik Etiketleme ve Membran Fraksiyonasyon<em&gt; Arabidopsis thaliana</em&gt; Süspansiyon Hücre Kültürleri

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Floresan Proteinler ifade Canlı Farelerde Görüntüleme hücrealtı Yapıların Intravital Mikroskopi

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

Görüntüleme Hücre Membran Hasarı ve Onarımı ilgilendim hücrealtı Süreçleri

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Yöntemler içinde Muscle hücrealtı Kompartmanları DeğerlendirecekÜSKÜP<em&gt; ° C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the spotlight and has been shown to have multiple major functions like actively modifying the extracellular matrix and forming an endocrine organ with the lacunocanalicular system that encloses it sending messages to distant sites. Owing to the methods that made it possible to test the osteocyte in vitro from isolating primary osteocytes to osteocyte-like cell lines, osteocytes are now experiencing a resounding interest and a surge of knowledge on structure and function. Many aspects of the osteocyte biology and interaction with other molecular components are yet to be discovered. In this protocol, we describe in detail the efficient isolation of primary osteocytes from dmp1-topaz neonatal mouse calvaria, which express the green fluorescent protein in osteocytes, through cell fractionation and subsequently acquiring cultures of primary osteocytes by FACS.

This protocol describes the dissection of neonatal dmp1-topaz mouse calvaria and isolation of osteocytes expressing the green fluorescent protein through cell digestion and fractionation, in addition to osteocyte preparation for fluorescence activated cell sorting (FACS).