Biz Zehavit Frenkel ve Sandra Tolchinsky teknik yardım için teşekkür ederim. Bu çalışma ile ilgili araştırmalar, İsrail Bilim Vakfı (1229-1207) ve Alman-İsrail Projesi İşbirliği (DIP-DFG) hibe tarafından destekleniyor.

Aşağıdaki protokol toplam glikoproteinler veya belirli bir glikoprotein ilgi şeker zincirleri analizleri için tasarlanmıştır. Prosedürü temelde birkaç değişikliklerle protokol boyunca belirtildiği gibi her iki durumda aynıdır. <ol><li> N-bağlı glukanlardır metabolik etiketleme için, bir gecede her bir örnek için 90 mm tabak içinde yetiştirilen memeli hücrelerinin bir akışkan kültürünü kullanmak için bir ihtiyacı farklı zaman noktalarında ya da tedavi temsil edebilir (numune). Bu protokol NIH 3T3 hücreleri için optimize edilmiştir. </li><li> 5 ml taze hazırlanmış% 10 diyaliz FCS ve 5-15 dakika için 4 mM sodyum piruvat ile birlikte önceden ısıtılmış (37 ° C) glukoz-orta, inkübasyon glikoz hücreleri açlıktan. </li><li> Açlık orta değiştirin önceden ısıtılmış 1 ml (37 ° C) glukoz-400 μCi içeren orta [2 - 3 H] (alt 65 konsantrasyonu μCi / ml Toplam glikoproteinler etiketlenmesi için kullanılan) mannoz-etiketli 1 saat için normal doku kültürü koşullarında hücrelerin kuluçkaya yatmaktadır. </li><li> Her örnekten etiketleme orta çıkarın ve dikkatle 4 2 ml PBS eklemek ° kovalamak için ilgili örnekleri gerekir sırasında nabız ve buz (bu örnekler bakliyat olarak anılacaktır) onları yere karşılık gelen numune C, 2 ml ile 3 kez durulanır önceden ısıtılmış (37 ° C), normal tam bir kültür ortamı ve daha sonra 37 CO 2 inkübatör yerleştirilir ° C, istenilen kovalamaca süreler için, önceden ısıtılmış düzenli orta 5 ml. </li><li> 2 ml buz gibi soğuk PBS ile darbe örnekleri (daha önce buz üzerinde yer) 3 kez durulayın. Bu hücreler daha sonra 2 ml PBS bir hücre kazıyıcı kullanarak çanak kazınmış ve 2 ml Eppendorf tüp yerleştirilir. </li><li> 16000Xg (6-10 sn) hücreleri Kısa-spin süpernatantı atmak ve -80 hücre pelletini ° C dondurmak </li><li> 5 ve kovalama, kovalamaca zaman sonunda numuneler için ayrıca 6 adımları gerçekleştirin. </li><li> Hücreleri dondurucuya kaldırın ve tampon 300 ul Ayrıca onlara lyse kısaca vorteks ve inkübasyon, tampon B ile buz üzerinde 20 dakika buz üzerinde 20 dk inkübe ya da toplam glikoproteinler analiz durumda, sonication tarafından takip (4 kez, 10 sn, maksimum genlik), sonra 5 dk örnekleri kaynatın. </li><li> 4 ° C&#39;de 20 dakika süreyle 16000Xg lizatları Santrifüj Süpernatantı yeni Eppendorf tüpüne aktarın ve pelet iptal. </li><li> Eğer toplam glikoproteinler analiz, moleküler filtrasyon ve deglycosylation (adım 17) geçin. H2A glikoprotein immunoprecipitation burada göstermiştir için, (Bizim örneğimizde H2A anti-antikor (Repligen), protein, A-agaroz boncuk 1:1 askı ve istenilen glikoprotein tavşan poliklonal anti H2A antikor 3 ul / numune 20l/sample eklemek ), adım 9 süpernatantı. </li><li> Yavaş dönüşü ile sürekli karıştırma, numuneler 4 ° C&#39;de 4-16 saat süreyle inkübe edin. </li><li> 30 saniye boyunca 16000Xg boncuk Spin, supernatant kullanarak vakum ve dikkatli bir şekilde çıkarın. </li><li> 500 ul tampon D ekleyerek ve vorteks, boncuk durulayın. Spin ve adım 12 gibi süpernatantı kaldırmak. Yıkama 3 kez tekrarlayın. </li><li> Boncuk pelet 10μl denatüre tamponu (NEB endo-H kiti ile birlikte) ekleyin ve 5 dakika (toplam proteinlerin analiz edildiğinde, deglycosylation Microcon filtresi, adım 17) örnekleri kaynatın. </li><li> (30 saniye için 16000Xg) boncuk aşağı çevirin ve yeni bir Eppendorf tüp içine süpernatantı transfer. Sonra, endo H enzim 0.5μl 10 ul reaksiyon tamponu (NEB endo H kiti ile birlikte) ile birlikte her örnek, örnekleri 37 ° C&#39;de 3 saat inkübe edilir. </li><li> Proteinlerden serbest glukanlardır ayırmak için, örnek sonra 3 dakika süreyle santrifüj 14000Xg, cut-off 30kDa 5 kez deiyonize su ile seyreltilir ve bir moleküler filtre (Microcon Ultracel YM30) üstüne konur. Deiyonize su numuneleri tekrar santrifüj 50μl uygulayın. Bu yıkanmaya flow-through tutarak, 2 kez daha tekrarlanır. </li><li> Toplam glikoproteinler analiz yapıyorsanız, Microcon filtre lizatları süpernatant (adım 9) geçerlidir. 14000Xg az 3 dakika santrifüj tekrarlanan 3 kez tampon E, 100 ul özü yıkayın. 10X endo H reaksiyon tamponu 3 ul ve Microcon retentate endo H enzim 1.5 ul ekleyin ve 37 az 3 saat boyunca inkübe ° C 3 tekrarlanan santrüfüj piyasaya glukanlardır adım 16 olarak deiyonize su ile yıkandı. </li><li> İsterseniz, H-endo dayanıklı glikoproteinler içeren 16 ve 17 adımları retentates, sonra 37, tampon C 15 ul N-glycosidase F 200mu inkübe ° C 16 saat Deiyonize su ile Elüsyon adım 16 olarak yapılır. </li><li> SpeedVac konsantratör endo H reaksiyon flow-through (adım 16 ve 17) içeren tüp YeriVe tamamen örnekleri kuru (bu 45 kadar yapılabilir ° C, bu süreci hızlandırmak için). </li><li> HPLC için numuneleri hazırlamak için, HPLC solvent (su, 60:40,% 1 fosforik asit asetonitril) 12 ul kuru pelet tekrar süspansiyon haline getirin. </li><li> HPLC cihazı (Spherisorb sütun bağlı) sabit çözücü akışı (1 ml / dk) ve basınç (1000 ve 2000 psi arasında belirli bir değer) ayarlayın ve her 1 dakikada bir tüp değiştirmek için bir fraksiyon toplayıcı yerleştirmek. </li><li> HPLC cihazı örnekleri Yük ve fraksiyon toplayıcı aynı anda başlatmak. </li><li> Her numune vadede ve standart bir oligosakkarit karışımı bir çalışma 1 ml 48 kesirler toplayın. </li><li> 500 ul Transferi, her fraksiyon bir sintilasyon flakon ve 3 ml su karışabilir sintilasyon sıvı içeriği karıştırın. </li><li> Şişeleri Yük ve sintilasyon sayacı okuma. </li><li> Kopyaya okuma kesir sayısı bir fonksiyonu olarak çizilir. </li><li> , Aşağıdaki gibi özel bir glukanıdır türler bir zirve içinde kopyaya değerleri temsil eder: kesirler 18-21 M8, M9 Kesirleri 36-39, M5, kesirler 22-26 ile M6, kesirler 27-31 M7, kesirler 32-35 karşılık kesirler G2M9 için 40-42 G1M9 ve kesirler 43-45. </li><li> Her tepe içinde kesirler için kopyaya değerleri toplamı, belirli bir glukanıdır türlerin mutlak miktarını yansıtır. Aslında her glukanıdır türün mutlak miktarı içerdiği mannoz kalıntılarının sayısına göre ayrılmıştır: daha mannoz kalıntılarını içeren türlerin daha fazla etiket, her glukanıdır türün aşağıdaki gibi hesaplanır &quot;göreli molar miktarı&quot; Satın aldığınız için telafi etmek için . Bu değer, daha sonra, her belirli glukanıdır türleri için, &quot;toplam yüzde&quot; elde etmek için elde edilen tüm glukanıdır türlerin göreceli molar miktarda toplamına bölünür. </li></ol><table border="1"><tbody><tr><td style="text-align:center;"> Chase (h) </td><td style="text-align:center;"> 0 </td><td style="text-align:center;"> 4 </td></tr><tr><td> Endo H (CPM) ile yayımlanan </td><td> 90000 </td><td> 16800 </td></tr><tr><td> N-glycosidase F (CPM) b ile Çıkış </td><td> 20400 </td><td> 3900 </td></tr><tr><td> Retentate (CPM) c de Glikoproteinler </td><td> 371700 </td><td> 127695 </td></tr><tr><td> Retentate (CPM) d Dolichol-oligosakkaritler </td><td> 4350 </td><td> 540 </td></tr><tr><td> Retentate Dolichol-oligosakkaritler (%) e </td><td> 1.4 ± 0.7 </td><td> 0.2 ± 0.2 </td></tr></tbody></table> Tablo 1: N-bağlı şeker zincirleri deglycosidases ve glikoprotein örneklerinde dolichol-oligosakkaritler varlığı serbest Analizi. NIH 3T3 hücreleri lizat darbe-kovalamaca prosedürü uygulanır. Microcon filtrasyon sonra, etiketli N-bağlı oligosakkaritler, darbe etiketleme sonra (yüksek mannoz tipi) ve N-glycosidase F karmaşık türü (Golgi işlenmiş glukanlardır) karşılık kovalamaca bir süre sonra daha ileri tedavi, endo H olmak üzere serbest bırakıldı endo H dayanıklı oligosakkaritler. Bu kantitatif analiz dolichol-oligosakkaritler, ilk retentate etiket önemsiz bir kısmını oluşturmaktadır belirlenir. Notlar: <ol class="lalpha"><li> NIH 3T3 hücreleri darbe [2 - 3 H] ile etiketlenmiş mannoz ve tam orta ile kovaladı. Microcon YM30 yoluyla hücre parçalama ve filtrasyon sonra, yüksek mannoz N-bağlı oligosakkaritler endo H. kuluçka tarafından retentates serbest bırakıldı </li><li> Endo (a) Microcon retentates H-dayanıklı malzeme daha sonra N-glycosidase F inkübe ve sintilasyon sayacı ölçülen oligosakkaritler serbest bırakıldı. </li><li> Metanol: su 10:10:03 habercisi glikolipitler kaldırmak ve 1N NaOH içinde çözünür ve bir sintilasyon sayacı sayılır unextracted malzeme Microcon retentates (a) endo H tedavi öncesi, kloroform ile 4 kez çıkarıldı olarak elde. </li><li> Dolichol-oligosakkaritler (c) 1 açıklanan ve sintilasyon sayacı sayılır alıntılar saflaştırıldı. </li><li> Ortalama retentate toplam cpm dolichol-oligosakkaritler (d) yüzde 3 deneyler (c + d). </li></ol> Temsilcisi Sonuçlar <img src="/files/ftp_upload/1899/1899fig1.jpg" alt="Şekil 1" /> Şekil 1. 1s sonra tedavi edilmezse hücreleri ve proteozomal inhibisyonu üzerine toplam NIH 3T3 glikoproteinler Pulse-kovalamaca analizi ile darbe etiketleme [2 - 3 H] biz bazı glucosylated beklenen bir profil eldeöncüleri (GLC 2 Man 9 GlcNAc 2 (G2M9) ve G1M9) kalan, fakat M9 içinde mevcut etiket ve M8 türlerin çoğu, ikincisi tüm glikoz kırparak ve bir mannoz kalıntısı (Şekil 1 A) sonucu. No ücretsiz mannoz veya diğer küçük öncüleri arınma titizlik doğrulayan tespit edildi. Oldukça uzun bir 8h kovalamaca ardından M7-6 ve M5 küçük bir miktar kırpma daha kapsamlı olduğunu, ancak en önemli türler, M9 ve M8 (Şekil 1 B) olmaya devam etmiştir. Kovalayan bir proteozomal inhibitörü (30 mcM MG-132) huzurunda yapıldı ise, bu aynı kesilmiş türlerin (Şekil 1 C) küçük bir birikim vardı. <img src="/files/ftp_upload/1899/1899fig2.jpg" alt="Şekil 2" /> Şekil 2. ERAD bir substrat şeker zincirleri Kantitatif analiz toplam glikoproteinler. (A) Şekil 1&#39;de birine benzer bir deneyde, her oligosakkarit türün bağıl molar miktarda mannoz içerik dayanarak hesaplandı. (B) Biz her glukanıdır tür için elde edilen kopyaya değerler dönüştürülür, sonra bütün türlerin mevcut bağıl molar miktarda toplamı göre her tür yüzde iki deneyler ortalama kovalamaca zamanın bir fonksiyonu olarak çizilen oldu. Benzer (A) ERAD substrat ASGPR H2A salınan bu glukanlardır hariç incelendi sonra varlığında 4h kovalamak için proteozomal inhibitörü MG-132 varlığı ya da yokluğu 4h için darbe etiketleme ve kovalamaca (C) Değerler (A) ve (B) ASGPR H2A ve glikoprotein havuzu için karşılaştırılır. MG-132 (D) N-bağlı şeker zinciri ER süreçleri kırparak Programı. Şeker kırpma işlemleri bu M9-8 karşılaştırıldığında ERAD hedeflenen proteinleri (R) Golgi ve ötesinde çıkın. Bağlantılı M6-5 yol Mannoz kalan 5-6 mannoz kalıntılarının tür verim N-glukanlardır kırparak ERAD süreci ile ilişkili olduğunu göstermektedir.

<ol>
	<li>Parodi, A. J., Behrens, N. H., Leloir, L. F., Carminatti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+polyprenol-bound+saccharides+as+intermediates+in+glycoprotein+synthesis+in+liver.">The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver.</a> Proc Natl Acad Sci U S A. 69 (11), 3268-3272 (1972).</li><li>Avezov, E., Frenkel, Z., Ehrlich, M., Herscovics, A., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+(ER)+mannosidase+I+is+compartmentalized+and+required+for+N-glycan+trimming+to+Man5-6GlcNAc2+in+glycoprotein+ER-associated+degradation.">Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation.</a> Mol Biol Cell. 19 (1), 216-225 (2008).</li><li>Frenkel, Z., Gregory, W., Kornfeld, S., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-associated+degradation+of+mammalian+glycoproteins+involves+sugar+chain+trimming+to+Man6-5GlcNAc2.">Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.</a> J Biol Chem. 278 (36), 34119-34124 (2003).</li><li>Hosokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+endoplasmic+reticulum+(ER)+degradation+of+misfolded+Null+Hong+Kong+alpha1-antitrypsin+by+human+ER+mannosidase+I.">Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.</a> J Biol Chem. 278 (28), 26287-26294 (2003).</li><li>Jakob, C. A., Burda, P., Roth, J., Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+misfolded+endoplasmic+reticulum+glycoproteins+in+Saccharomyces+cerevisiae+is+determined+by+a+specific+oligosaccharide+structure.">Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.</a> J Cell Biol. 142 (5), 1223-1233 (1998).</li><li>Tolchinsky, S., Yuk, M. H., Ayalon, M., Lodish, H. F., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+versus+secreted+forms+of+human+asialoglycoprotein+receptor+subunits.+Role+of+a+juxtamembrane+pentapeptide.">Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide.</a> J Biol Chem. 271 (24), 14496-14503 (1996).</li></ol>

HPLC ayırma ile canlı hücrelerin içinde glukanlardır darbe-kovalamaca analiz oligosakkarit yapısal değişikliklerin dinamikleri bir glikoprotein ömrü boyunca çalışmak için bir yöntem sağlar. ER katlama, kalite kontrol ve ticareti sistemleri 2-5 sinyalleri üreten bu tip değişikliklerin yer aldığını giderek artan kanıtlar vardır. Bu yöntem değil, sadece belirli bir ilgi glikoprotein için değil, aynı zamanda biz belirli bir ERAD substrat zıt kader ve hücresel bir glikoprotein havuz...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>600E Multisolvent delivery System controller</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT062710</td>
<td>&nbsp;</td>
<tr>
<td>Acetonitrile LiChrosolv (gradient grade for liquid chromatography)</td>
<td>&nbsp;</td>
<td>Merck Bioscience</td>
<td>1.0003</td>
<td>&nbsp;</td>
<tr>
<td>Microcon Amicon Ultra 0.5 ml 30K or Centricon ultracel YM-30</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC503024 or 4208</td>
<td>&nbsp;</td>
<tr>
<td>Concentrator 5301, incl. 48 x 1.5 / 2.0 ml fixed-angle rotor</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5301 000.016</td>
<td>&nbsp;</td>
<tr>
<td>Dialyzed Foetal Calf Serum</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-011-1A</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modi&#xFB01;ed Eagle's Medium</td>
<td>&nbsp;</td>
<td>Gibco Invitrogen Cell Culture</td>
<td>41965039</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Modi&#xFB01;ed Eagle's Medium - glucose free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D5030</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>EDTA disodium salt (Titriplex Iii)</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>1084180250</td>
<td>&nbsp;</td>
<tr>
<td>Endo Hf Kit (1,000,000 units/ml)</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>p0703S</td>
<td>&nbsp;</td>
<tr>
<td>Foetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-001-1A</td>
<td>&nbsp;</td>
<tr>
<td>Frac-100 fraction collector</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>18-1000-77</td>
<td>&nbsp;</td>
<tr>
<td>LS 6500 Liquid Scintillation Counting systems</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>510720</td>
<td>&nbsp;</td>
<tr>
<td>Mannose, D-[2-3H(N)]- (Specific Activity: 15-30Ci/mmol)</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>NET570A</td>
<td>&nbsp;</td>
<tr>
<td>N-carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>474790</td>
<td>&nbsp;</td>
<tr>
<td>N-glycosidase F (1000 units/ml)</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11365177</td>
<td>&nbsp;</td>
<tr>
<td>N-octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>O3757</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 cells</td>
<td>&nbsp;</td>
<td>American Type Culture Collection (ATCC)</td>
<td>CRL-1658</td>
<td>&nbsp;</td>
<tr>
<td>Opti-Flour</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>6013199</td>
<td>&nbsp;</td>
<tr>
<td>Phosphoric acid solution ( 49-51%, for HPLC)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>79607</td>
<td>&nbsp;</td>
<tr>
<td>Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2714</td>
<td>&nbsp;</td>
<tr>
<td>Protein A-Sepharose beads</td>
<td>&nbsp;</td>
<td>Repligen</td>
<td>IPA300</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit polyclonal anti-H2 carboxy-terminal 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>30970</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Bio-RAD</td>
<td>161-0301</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>342483</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution (100mM)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>Spherisorb NH2 Column, 5 &mu;m, 4.6 x 250 mm</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>PSS831115</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>BDH</td>
<td>306324N</td>
<td>&nbsp;</td>
<tr>
<td>Vibra-Cell ultrasonic processors VCX 750</td>
<td>&nbsp;</td>
<td>Sonics and Materials, Inc.</td>
<td>690-003</td>
<td>&nbsp;</td>
<tr>
<td>Buffer A: 1% Triton X-100, 0.5% w/v sodium deoxycholate, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer B: 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer C: N-glycosidase F reaction buffer: 200mM Na3PO4, pH 8.0, 4mM EDTA, 1.5% N-octylglucoside</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 stably expressing H2a 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer D: 0.5% Triton X-100, 0.25% w/v sodium deoxycholate, 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer E: 0.5% w/v SDS, 1% v/v 2-Mercaptoethanol, Protease inhibitor cocktail 2% v/v in PBS </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M3148</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

Eklenti, GLC<sub&gt; 3</sub&gt; Erkek<sub&gt; 9</sub&gt; GlcNAc<sub&gt; 2</sub&gt; ER doğmakta olan polipeptidlerin oligosakkarit habercisi sekretuar proteinler için ortak bir değişiklik. Bu değişiklik, çeşitli biyolojik süreçlerde sorumlu olmasına rağmen, onun işlevini glikoprotein kalite kontrol ve ticareti için sinyallerinin üretimi rolünü son kanıtlar, ek yönlerini ortaya çıkmaktadır. Böylece, N-bağlı glukanlardır ve işleme ilgili fenomenler yoğun araştırmalar yapılmaktadır. Proteomik analiz için son zamanlarda geliştirilen Yöntemleri glikoprotein N-bağlantılı glukanlardır karakterizasyonu büyük ölçüde düzeldi. Buna rağmen, şeker zinciri işleme dahil dinamikleri bilgiler sağlayabilir yok. Bunun için, etiketleme ve darbe-kovalamaca analiz protokolleri genellikle karmaşık ve çok düşük verim vermesi kullanılır. Biz burada metabolik etiketli N-bağlı oligosakkaritler izolasyonu ve analizi için basit bir yöntem açıklanmaktadır. Protokol [2 hücre etiketleme dayalı.<sup&gt; 3</sup&gt; H] ilgi ya özellikle immunoprecipitated bir protein veya moleküler filtrasyon (Amicon) takip endo H ve N-glycosidase F ardışık tedaviler, genel glikoprotein havuzu mannoz, denatüre lizis ve oligosakkaritler enzimatik serbest. Bu yöntemde izole oligosakkaritler tek bir monosakkarit bir çözünürlüğe sahip, HPLC analizi, onları oluşan monosakkarit ünitelerinin sayısına göre çeşitli glukanıdır yapılar arasında ayrımcılık sağlar için bir giriş olarak hizmet vermektedir. Bu yöntemi kullanarak, normal şartlarda darbe-kovalamaca sonra proteazom inhibisyonu altında toplam hücre glikoproteinler yüksek mannoz N-bağlı oligosakkarit profilleri çalışma başardık. Bu profiller bir immunoprecipitated ER-ilişkili bozulması (ERAD) substrat elde edilen karşılaştırıldı. Bizim en NIH 3T3 hücresel glikoproteinler nispeten istikrarlı olduğunu ve kendi oligosakkaritler Man için kesilmiş olduğunu göstermektedir<sub&gt; 9-8</sub&gt; GlcNAc<sub&gt; 2</sub&gt;. Buna karşılık, kararsız ERAD substratlar Man kesilmiş.<sub&gt; 6-5</sub&gt; GlcNAc<sub&gt; 2</subBu türlerin taşıyan ve glikoproteinler proteozomal bozulması inhibisyonu üzerine birikir.

Watch this Scientific Journal Video about Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells at JoVE.com

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Biz, memeli hücrelerinde biyosentezi sonra glikoproteinler erken ömrü boyunca N-bağlı glukanlardır değişiklik analizi için bir yöntem açıklanmaktadır. Bu darbe-kovalamaca metabolik etiketli glukanlardır, glikoproteinler ve HPLC ile incelenmesi enzimatik serbest analizi ile elde edilir.

Memeli Hücrelerinde Glikoproteinler N-bağlı Şeker Zincirler Pulse-kovalamaca Analizi

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

pulse-chase-analysis-n-linked-sugar-chains-from-glycoproteins

Research

JoVE Journal

Biology

15.1K Görüntüleme.  Tel Aviv University. Biz, memeli hücrelerinde biyosentezi sonra glikoproteinler erken ömrü boyunca N-bağlı glukanlardır değişiklik analizi için bir yöntem açıklanmaktadır. Bu darbe-kovalamaca metabolik etiketli glukanlardır, glikoproteinler ve HPLC ile incelenmesi enzimatik serbest analizi ile elde edilir.

Video: Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

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, ...

Biyoloji

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

Memeli Hücrelerde DNA çift iplikli Break (DSB) Tamir Analizi

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

Memeli Hücreleri RNAi Tarama MİSYON esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

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 ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Mikroenjeksiyon tarafından Memeli Hücreleri mRNA Nükleer İhracat Kinetik Analizi

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Memeli Hücreler, Hücre Döngüsü Pozisyon Analizi

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Memeli Linker-histon Alttipleriyle İfadesi Analizi

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Memeli hücrelerinde endoplazmik retikulum lokalize mRNA&#39;ların görselleştirilmesi

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...