Bu çalışmada kullanılan C. elegans suşları Ulusal Sağlık Enstitüleri tarafından - Office of araştırma altyapı programları (P40 OD010440) desteklenen Caenorhabditis genetik Merkezi'nden elde edilmiştir. Bu eser NIH tarafından desteklenmiştir: 1R03AG048578-01 Jun Liang, Jun Liang ve PSC CUNY-CCRG 1501-CUNY 66184-00 44 ve 67248-00 45 Jun Liang için. Cathy Savage-Dunn nazikçe onu laboratuvar alanı paylaşımı için teşekkür ederiz. Tüm floresans görüntüleri Queens Koleji çekirdek tesisinde toplanmıştır. William J. Rice el yazması üzerine onun yorum için teşekkür.

1. çözümler
<ol><li>
NGM plakaları
	<ol>
<li>Bir 2 L Erlenmeyer şişesi, NaCl, 3 g, agar 17 g, pepton 2.5 g ve dH2O. kapak alüminyum folyo ile balonun ağzı 975 mL ekleyin. Otoklav şişesi 50 dk. 20-30 dakika sakin ol.</li>
		<li>Sonra steril çözümleri ekleyin: 1 mL 1 M CaCl2, 5 mg/mL kolesterol etanol içinde 1 mL, 1 M MgSO41 mL ve 1 M KPO4 (pH 6.0) arabelleği 25 mL. Onları iyice karıştırın çözümleri girdap.</li>
		<li>Sıvı Sabunluk kullanarak, 60 mm Petri plakaları NGM çözümü dağıtmak. Ağar kaplamalar 2/3 tam dolgusuna. Onları ileride kullanmak üzere 4 ° C'de hava geçirmez bir kap içinde saklayın.</li>
	</ol>
</li>
	<li>
1 M KPO4 arabellek (pH 6.0)
	<ol>
<li>KH2PO4 10.83 g ve 3.56 g K2HPO4 ' te dH2O çalışma geçiyoruz, o zaman 100 ml toplam ses seviyesini. Basınçlı kap 15dk için çözüm.</li>
	</ol>
</li>
	<li>
M9
	<ol>
<li>3 g KH2PO4, 6 g Na2HPO dağıtılması4ve 5 gram NaCl dH2O, 1 M MgSO41 mL ekleyin ve 1 m'ye toplam ses seviyesini</li>
	</ol>
</li>
	<li>
10 µg/mL DAPI
	<ol>
<li>10 mg/ml DAPI dH2O. 999 µL içine 1 µL Ekle</li>
	</ol>
</li>
	<li>
% 95 alkol (v/v)
	<ol>
<li>95 mL %100 alkol ölçmek ve ses seviyesini 100 ml distile su ekleyin.</li>
	</ol>
</li>
	<li>
% 30 aseton (v/v)
	<ol>
<li>30 mL aseton dH2için O ekleyin ve toplam ses seviyesini 100 ml.</li>
	</ol>
</li>
</ol>2. ısı stres
<ol><li>Hedef genlerin9,10translasyonel bir yapı ile bir extrachromosomal dizi satırı oluşturmak. Alternatif olarak, bu satırları üzerinden Caenorhabditis genetik Merkezi ( C. elegans araştırma için bir kamu kaynağı olan CGC), veya daha önce yayımlanmış araştırma laboratuvarı alabilirsiniz.</li>
	<li>Extrachromosomal satırları solucanlar 3.800 Rad γ-radyasyon9açarak genom entegre. Sonra her hayvan bir işaretleyici protein GFP veya silindir gibi ifade eder stabil ifade çizgileri seçin.</li>
	<li>Radyasyon sırasında oluşturulan mutasyonlar kaldırmak için çizgiler en az 2 outcross x. Protein ifade deseni değişiklikleri stres altında transgenik bu cümleyi.</li>
	<li>NGM solucan plakaları 1.1. adımda açıklandığı şekilde hazırlayın. Bir OP50 klon çizgi ve sıvı LB suyu bakterilerde gecede kültür. Solucanlar için bir besin kaynağı olarak bir bakteri çim büyümeye sıvı OP50 NGM pilakalar kültür ve 37 ° C kuluçka plakaları kuluçkaya tohum gecede. Plakalar için kullanmadan önce en az 30 dakika oda sıcaklığında sakin.</li>
	<li>20 ° C ısı stres tahlil öncesinde en az 2 nesiller için açlık olmadan transgenik satırında büyümek.</li>
	<li>8-10 genç gravid yetişkin (rahim yumurta ile dolu) bir yeni solucan plaka, sonra onları yumurtlamak için yaklaşık 3-5 h let için almak ve tüm yetişkin plakalar kaldırın.</li>
	<li>Solucanlar eşitlemek için yumurta yumurtadan ve larvaları yaklaşık 48 saat için dördüncü larva (L4) sahne alanı'na büyümeye izin. L4 sahne (Şekil 1, ok)11tanımlamak için onların vulva yapısı (yarım ay yapısı) inceleyin.</li>
	<li>Isı stres ulaşmak 2-5 h için 35 ° C kuluçka L4 larvaları ile solucan tabak koyun. Bir termometre doğru ısısını ölçmek için kuluçka makinesine koyun.</li>
</ol>3. DAPI boyama
<ol><li>
Etanol fiksasyon
	<ol>
<li>10 µL M9 arabelleği bir cam slayt ortasına ekleyin.</li>
		<li>Isı şok solucanlar adım 2.8 toplar ve onları koyardım M9 arabelleğine cam slayt üzerinde.<ol>
<li>Alternatif olarak, M9 ile plaka uzakta yıkama, 1000 x g oda sıcaklığında 1 dk. için de santrifüj kapasitesi ve süpernatant atmak. Solucanlar için cam slayt ekleyin.</li>
		</ol>
</li>
		<li>Herhangi bir aşırı sıvı boşaltmak için yumuşak bir doku kullanın. Diseksiyon mikroskop altında bu adıma dikkat et.</li>
		<li>10 µL % 95 alkol solucanlar üzerine eklemek ve onları kurutma. Diseksiyon mikroskop altında süreci izlemek ve çok kuru olsun hayvan izin vermeyin. Sonra hemen etanol hayvanlardan buharlaşıp vardır, sonraki adıma geçin. Not: Etanol çok hızlı buharlaşır.</li>
		<li>Adımı yineleyin 3.1.4 iki kez böylece solucanlar sabit. Not: Bu hayvanlar için karanlık ve çarpık bakmak normaldir.</li>
		<li>DAPI 10 µL ekleyin (10 µg/mL) solucanlar için. Hemen onları bir coverslip ile karşılamak ve şeffaf oje jel içeren slaydı mühür.</li>
		<li>Floresan mikroskop üzerinde yaklaşık 10 dakika sonra hayvanlar görüntüleyin.</li>
	</ol>
</li>
	<li>
Aseton fiksasyon (alternatif bir yaklaşım)
	<ol>
<li>Adım 2.8 M9 ile ısı şok plaka uzakta yıkama, 1000 x g oda sıcaklığında 1 dk. için de santrifüj kapasitesi ve süpernatant atmak. Pelet dH2O 1 mL ile yıkayın, Pelet toplamak ve süpernatant atın.</li>
		<li>1000 x g için oda sıcaklığında 1 dk 400 µL % 30 aseton 15 dk. santrifüj ekleyin ve süpernatant atın. Hayvanlar 2 yıkamak için dH2O 500 µL kullanın x. Süpernatant atmak.</li>
		<li>DAPI 200 µL ekleyin (10 µg/mL), veya 4 x tüp 15 dakika içinde toplam solucanlar birim miktarı.</li>
		<li>1000 x g 1 dk. için de santrifüj kapasitesi ve süpernatant atın. Pelet 2 yıkama x 500 µL dH2O. ile solucanlar cam slaytlarda yer, onları coverslips ile karşılamak, şeffaf oje jel içeren slaydı mühür ve floresan mikroskop altında hayvanlar gözlemlemek.</li>
	</ol>
</li>
</ol>4. mikroskobik görüntüleme
<ol><li>UV ışık kaynağı ve floresan mikroskop açın. Mikroskop için bağlı olduğu bilgisayarda açın.</li>
	<li>Floresan mikroskop slaytta yükleyin. Solucanlar bulmak, 400 X büyütme gücünü arttırın ve numune üzerinde odaklanmak için bir düşük güç objektif lens (10 X) kullanın.</li>
	<li>Mikroskop ile sağlanan yazılımını açın. "Alma" menüsünde'ı tıklatın; "Kamera" tıklayın ve mikroskop için bağlı kamera seçin; Bu yazılım ile iletişim kurmak seçili kamera sağlar. Not: Farklı mikroskoplar işleminde değişiklik gösterebilir.</li>
	<li>Altında aynı "alma", "Çok boyutlu edinme"'ı tıklatın. Bu yeni bir "Çok boyutlu edinme" gezinti penceresi açar. "Çok kanallı" düğmesini ve tüm kanallar görüntülenir.</li>
	<li>Mavi, yeşil ve numune gözlemlemek için DIC (diferansiyel girişim kontrast) seçin. "Çok boyutlu edinme" Gezinti penceresinde "Mavi-DAPI", "Yeşil-GFP" ve "Gri-DIC", "Nomarski" kanallarda bırakın; "Kırmızı-rodamine" ve "beyaz parlak alan", gibi diğer kanallar sağ tıklayın onları kapatın.</li>
	<li>
İlk olarak, pozlama süresi her kanal için tedbir:
	<ol>
<li>Seçin ve sonra otomatik çekim hızı ile DAPI kanalın altında canlı bir görüntü çekmek için "Ölçü" tıklayın "Mavi-DAPI" kanal sol tıklatın. Canlı bir görüntü ile yeni bir pencere açılacaktır.</li>
		<li>Canlı görüntü penceresinin sol tarafında el ile istediğiniz gibi resim yapmak gerekirse çekim hızı ayarlayın.</li>
		<li>Son olarak "Tamam tıklayın" canlı görüntü penceresinde. Bu çekim hızı DAPI kanalın altında ayarlar.</li>
	</ol>
</li>
	<li>Diğer kanallar, "Yeşil-GFP" ve "Gri-pozlama süreleri bu kanallar için ayarlamak için DIC", "Nomarski" için 4,6 arasındaki adımları yineleyin.</li>
	<li>"Çok boyutlu edinme" Gezinti penceresinde otomatik olarak 3 resim sipariş (bkz. yukarıda) belirtildiği gibi belirli pozlama süreleri ile 3 farklı kanalların altında toplamak için "Başlat" tıklayın.</li>
	<li>Kurtarmak belgili tanımlık eğe için ana menüye gidin, "Dosya" ve sonra "Kaydet"'i tıklatın. Dosya adını ve klasör adını girin. İleride detaylı görüntüleme bilgileri korumak için varsayılan dosya biçimi olarak kaydedin.</li>
	<li>
Diğer biçimlerde dosyasını dışa aktarmak için:
	<ol>
<li>Gitgidmek "Eğe" ve tıkırtı üstünde "ihracat yapmak". Bu bir dışa aktarma ayarı pencere açılacaktır.</li>
		<li>Dosya adı ve klasör ayarlayın. "Bir proje klasörü için daha iyi oluşturmak" verileri düzenlemek kontrol edin. Yazılım aynı zamanda 4 diğer seçenekleri belirtmek kullanıcı sağlar: "Generate birleştirilmiş görüntüler"kanal görüntülerde renk kullanın",", "Kanal adları kullan" ve "Yalnızca birleştirilmiş resim".</li>
		<li>Açılan menüden "TIF", "BMP", "PSD", veya "JPG" ve diğer türleri gibi istediğiniz dosya türünü seçin. O zaman tıkırtı üstünde "ihracat Başlat".</li>
	</ol>
</li>
</ol>

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J., Shen, Z. Z., Cunha, A., Emmons, S. W., Leroi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+polyploidization+and+cellular+proliferation+drive+body+size+evolution+in+nematodes.">Somatic polyploidization and cellular proliferation drive body size evolution in nematodes.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (10), 5285-5290 (2000).</li><li>Nagamatsu, Y., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+the+control+of+body+size+by+a+G-kinase+and+a+downstream+TGFbeta+signal+pathway+in+Caenorhabditis+elegans.">Mechanisms for the control of body size by a G-kinase and a downstream TGFbeta signal pathway in Caenorhabditis elegans.</a> Genes to Cells. 9 (1), 39-47 (2004).</li></ol>

Yazarlar bildirmek için bir şey yok.

Bu makale için çekirdek sitoplazma protein hücre altı değişiklikleri doğrulamak için hızlı ve etkili bir yöntem mevcuttur. Çekirdek yapı (Şekil 3) boyama DAPI tarafından doğrulandı ise protein ifade GFP füzyon tarafından gösterildi. İmmunostaining C. elegans proteinler zor olduğundan, çoğu C. elegans protein hücre altı yerelleştirmeler GFP, Laz, mCherry ve diğerleri gibi işaret proteinler ile etiketleyerek karakterizedir18</sup...

Protein hücre altı yerelleştirme değişikliği yanıt olarak dahili veya harici sinyaller ısı stres, açlık, oksidatif stres, apoptozis, protein fosforilasyon ve diğerleri gibi ortak bir mekanizmadır. Örneğin, ısı stres FOXO üye DAF-16 nükleer translocation1,2ve3,4sinyal alıcı ölümü üzerine mitokondri için teklif translocates pro-apoptotik BCL-2 protein neden olmaktadır. Çeşitli teknikler bu değişiklikleri algılamak kullanılabilir. Western blot ve biyokimyasal olarak izole hücre altı yapıları (Örneğin, mitokondri veya çekirdek) bir arada de hedef3elde edebiliriz. Ancak, spesifik bir antikor protein ilgi karşı gerektirir. Böylece, köklü bir antikor başarı için anahtar olur. Farklı hücre altı yapıları veya organelleri yeşil floresans protein (GFP), kırmızı floresans protein (RFP), sarı floresans protein (YFP) ve mCherry gibi çeşitli işaretçileri olan etiket ve bu arada protein etiket için alternatif bir yaklaşımdır ilgi diğer işaretleri ile. O zaman, onları hedef5,6yerelleştirmek için confocal mikroskop altında gözlemlemek. Radyoaktif izotoplar hedef proteinler etiketleme ve onların hücre altı yerelleştirme7algılama için alternatif bir seçimdir. Ancak, bu yöntem uygun eğitim ve radyoaktif atıkların işlenmesi gerekir. Spesifik bir antikor eksikliği, uygun bir işaretleyici yokluğu veya ekipman confocal mikroskop gibi scarceness gibi bu şartlar altında başka bir yaklaşım dikkate alınması gerekiyor. Protein nükleer translocation tanımlamak için sadece bir işaretleyici ile hedef proteinler etiketlemek için ve çekirdeklerin Kimyasal reaktif 4', 6-diamidino-2-phenylindole (Bu sadece normal floresan mikroskop gerektirdiğinden DAPI) ile leke için çekici.
Immunolabeling C. elegans antikorlar ile yumurta kabuğu veya hayvan çevreleyen kolajen kütikül düşük geçirgenliği nedeniyle meydan okuyor. Bu arada, C. elegans proteinler--dan onların omurgalı orthologs önemli ölçüde farklı olduğu için birkaç ticari şirketler ile belirli ürünler C. elegans sağlar. C. elegans antikorlar kendileri için üretmek küçük bir laboratuvar için zordur. Araştırmacılar toplumda sık sık tagged proteinler protein yerelleştirme veya gen ifade göstermek için imleyicileri kullanın. Bu makale EXL-1::GFP bir protein nükleer translocation ısı stres8altında izlemek için örnek olarak kullanılmıştır. Bir entegre translasyonel exl-1::gfp içine hayvan genom stabil gen gfp füzyon ile ifade etmek için kullanılır. Araştırma exl-1 bağırsak, vücut duvar kas ve diğer hücre altı yapıları8ifade edilir gösterdi. Bu iletişim kuralı, solucanlar dördüncü larva (L4) sahneye eşleştirir, ısı stres deney ve davranış DAPI boyama, etanol fiksasyonu, aseton fiksasyon ve görüntüleme düzenli floresan mikroskop altında gerçekleştirmek gösterilmiştir.

<table>
	<tbody>
		<tr>
			<td>DAPI</td>
			<td>Biotium</td>
			<td>40043</td>
			<td>both Hoechst 33258 and Hoechst 33342 also works well</td>
		</tr>
		<tr>
			<td>OP50</td>
			<td>CGC</td>
			<td>OP50</td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Kim Wipe</td>
			<td>Kimberly-Clark Corporation</td>
			<td></td>
			<td>soft tissue</td>
		</tr>
		<tr>
			<td>Zeiss&#160; AX10</td>
			<td>Zeiss</td>
			<td></td>
			<td>fluorescence microscope</td>
		</tr>
		<tr>
			<td>Axiovision Rel 4.8</td>
			<td>Zeiss</td>
			<td></td>
			<td>microscope software</td>
		</tr>
		<tr>
			<td>AxioCam MR Rev3</td>
			<td>Zeiss</td>
			<td></td>
			<td>digital camera</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent nail polish gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-20 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-200 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum wire worm pick</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans

Bu iletişim kuralı, bir yeşil floresans protein (GFP) füzyon protein ve 4', 6-diamidino-2-phenylindole (DAPI) boyama protein hücre altı yerelleştirme değişiklikleri; izlemek için kullanılır Özellikle, bir ısı altında nükleer translocation stres durumu. Proteinler buna bağlı olarak dış ve iç sinyalleri tepki. Onun hücre altı yerelleştirme değiştirmek için ortak bir mekanizmadır. Bu makalede bir antikor, radyoaktif etiketleme veya confocal mikroskop gerektirmez protein yerelleştirme izlemek için bir iletişim kuralı. Bu makalede, GFP memeli CLIC4 de dahil olmak üzere klorür hücre içi kanal proteinleri (CLICs) ailesinin C. elegans, üye EXL-1 hedef protein etiketlemek için kullanılır. Bir entegre translasyonel exl-1::gfp transgenik satır (bir organizatörü ve tam gen sıralaması) dönüştürme ve γ-radyasyon tarafından oluşturulmuş ve stabil gfpve gen ifade eder. Son araştırma ısı stres, değil oksidatif stres, EXL-1::GFP çekirdeğinde biriken gösterdi. GFP sinyal çekirdek yapısı ve DAPI ile örtüşen sinyalleri stres altında EXL-1 hücre altı yerelleştirme değişiklikleri onaylar. Bu iletişim kuralı DAPI boyama için iki farklı fiksasyon yöntemi sunar: etanol fiksasyon ve aseton fiksasyon. Bu makalede sunulan DAPI boyama iletişim kuralı hızlı ve verimli ve GFP sinyal hem protein hücre altı yerelleştirme değişiklikleri korur. Bu yöntem yalnızca Nomarski, FITC filtre ve DAPI filtre ile floresan mikroskop gerektirir. Bir küçük laboratuvar ayarı, lisans öğrencisi araştırma, lise öğrenci araştırma ve biyoteknoloji derslik için uygundur.

Klorür hücre içi kanal proteinler (klik) türler12arasında son derece korunmuş çok fonksiyonlu proteinlerdir. CLICs hücresel stres, autophagy, apoptozis, karsinojenezis, anjiogenez ve makrofaj doğuştan gelen bağışıklık yanıtı memeli sistem13,14,15,16 düzenleyen çok araştırma gösterir ,17<...

Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Bu iletişim kuralı bir protein nükleer translocation ısı stres altında bir yeşil floresans protein (GFP) füzyon proteini bir işaretleyici olarak ve 4', 6-diamidino-2-phenylindole (DAPI) kullanarak izlemek gösterilmiştir boyama. İletişim kuralı boyama DAPI hızlı ve GFP ve protein hücre altı yerelleştirme sinyalleri korur.

Protein hücre altı yerelleştirme yeşil floresans Protein etiketleme ve 4', 6-Diamidino-2-phenylindole Caenorhabditis elegans içinde boyama tarafından tespit

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

detecting-protein-subcellular-localization-green-fluorescence-protein

Research

JoVE Journal

Biology

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

9.5K Görüntüleme.  Borough of Manhattan Community College/CUNY. Bu iletişim kuralı bir protein nükleer translocation ısı stres altında bir yeşil floresans protein (GFP) füzyon proteini bir işaretleyici olarak ve 4', 6-diamidino-2-phenylindole (DAPI) kullanarak izlemek gösterilmiştir boyama. İletişim kuralı boyama DAPI hızlı ve GFP ve protein hücre altı yerelleştirme sinyalleri korur.

Video: Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Temel<em&gt; Caenorhabditis elegans</em&gt; Yöntem: Senkronizasyon ve Gözlem

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

MBP tabanlı Etiketleme Stratejisi Mgm101 Rekombinasyon Protein hazırlanması

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Tütün protoplastlarında ve Yapraklar Bimoleküler Floresan tamamlama tarafından Visualized protein-protein etkileşimleri

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Membran proteinlerinin Yeşil floresan proteini temelli Ekspresyon elemesinde<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Çoklanmış Immuno-histokimyasal boyanması ve Multispektral Görüntüleme kullanma Protein Ekspresyonu ve Co-lokalizasyon Kantifikasyonu

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

Floresans Merkezi kütlesi Imaging tarafından Golgi protein nicel yerelleştirme

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Yöntemleri ile Caenorhabditis elegans çağda doğal Protein toplama çalışma değişikliklere

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Protein hücre altı yerelleştirme yeşil floresans Protein etiketleme ve 4', 6-Diamidino-2-phenylindole Caenorhabditis elegans içinde boyama tarafından tespit

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