Ringraziamo Zehavit Frenkel e Sandra Tolchinsky per l&#39;assistenza tecnica. Ricerca nel settore di questo lavoro è supportato anche da finanziamenti la Israel Science Foundation (1229-1207) e il tedesco-israeliano di cooperazione del progetto (DIP-DFG).

Il seguente protocollo è stato progettato per l&#39;analisi delle catene di zuccheri di glicoproteine ​​totale o di una glicoproteina di interesse specifico. La procedura è sostanzialmente la stessa per entrambi i casi con alcune modifiche come affermato in tutto il protocollo. <ol><li> Per l&#39;etichettatura metabolico di N-glicani legati uno ha bisogno di usare una cultura subconfluent di cellule di mammifero coltivate durante la notte in un piatto da 90 mm per ogni campione (i campioni possono rappresentare diversi momenti o trattamenti). Questo protocollo è ottimizzato per le cellule NIH 3T3. </li><li> Affamare le cellule di glucosio da parte incubazione in 5 ml di preparati al momento pre-riscaldato (37 ° C) glucosio senza media, in dotazione con il 10% FCS dializzati e 4 mM di piruvato di sodio per 5-15 minuti. </li><li> Sostituire il supporto fame con 1 ml di pre-riscaldato (37 ° C) glucosio senza supporto contenente 400 μCi di [2 - 3 H]-marcato mannosio (la concentrazione più bassa di 65 μCi / ml viene usato per l&#39;etichettatura di glicoproteine ​​totale), e incubare le cellule in condizioni normali coltura dei tessuti per 1 ora. </li><li> Rimuovere il supporto di etichettatura da ogni campione, ed aggiungere con cautela 2 ml di PBS a 4 ° C per i campioni corrispondenti a impulsi e disporli sul ghiaccio (questi campioni sono indicati come legumi), mentre i campioni corrispondenti alla caccia devono essere risciacquati 3 volte con 2 ml di pre-riscaldato (37 ° C) media normale cultura completa, e quindi posto in un incubatore CO 2 a 37 ° C con 5 ml di pre-riscaldato medio regolare per i periodi di caccia desiderato. </li><li> Sciacquare i campioni di impulsi (precedentemente immessi sul ghiaccio) 3 volte con 2 ml di PBS ghiacciata. Le cellule sono poi raschiato il piatto in 2 ml di PBS usando un raschietto cellula, e messo in una provetta da 2 ml Eppendorf. </li><li> Short-spin le cellule a 16000Xg (6-10 sec), scartare il supernatante e congelare il pellet di cellule a -80 ° C. </li><li> Eseguire i punti 5 e 6 anche per i campioni inseguimento al termine del tempo di caccia. </li><li> Rimuovere le cellule dal congelatore e lisare loro con l&#39;aggiunta di 300 ml di tampone A, vortex brevemente e incubando per 20 minuti sul ghiaccio, o nel caso di totale analisi glicoproteine, mediante incubazione 20 min in ghiaccio con tampone B, seguita dalla sonicazione (4 volte, 10 sec, massima ampiezza), poi far bollire i campioni per 5 minuti. </li><li> Centrifugare i lisati a 16000Xg per 20 minuti a 4 ° C. Trasferire il surnatante in una nuova provetta Eppendorf e scartare il pellet. </li><li> Se l&#39;analisi di glicoproteine ​​totale, procedere alla filtrazione molecolare e deglycosylation (punto 17). Per immunoprecipitazione della glicoproteina H2a dimostrato qui, aggiungere 20l/sample di (Repligen) proteina A-agarosio perline 01:01 sospensione e 3 ml / campione del nostro coniglio policlonale anticorpo anti H2a per la glicoproteina desiderato (nel nostro esempio anticorpi anti-H2a ), per il surnatante dal punto 9. </li><li> Incubare i campioni per 4-16 ore a 4 ° C, mescolando continuamente con rotazione lenta. </li><li> Spin giù le perle al 16000Xg per 30 secondi, e rimuovere con attenzione il vuoto surnatante utilizzando. </li><li> Sciacquare le perline, con l&#39;aggiunta di 500 microlitri di buffer D e vortex. Spin, come nel passaggio 12 e rimuovere il surnatante. Ripetere il lavaggio per 3 volte. </li><li> Aggiungere 10μl di buffer denaturazione (fornito con il endo-H NEB kit) al pellet tallone e bollire i campioni per 5 minuti (quando le proteine ​​totali sono analizzati, il deglycosylation viene eseguita sul filtro microcontrollore, punto 17). </li><li> Spin giù le perline (16000Xg per 30 secondi), e trasferire il surnatante in un nuovo tubo Eppendorf. Poi 0.5μl di enzima endo H vengono aggiunti ad ogni campione con 10 ml di buffer di reazione (anche in dotazione con il kit endo ONA H), ei campioni sono incubati a 37 ° C per 3 ore. </li><li> Per separare i glicani rilasciati dalle proteine, il campione viene diluito 5 volte con acqua deionizzata e posto sulla cima di un filtro molecolare (microcontrollore Ultracel YM30), con un 30kDa cut-off, poi centrifugato a 14000Xg per 3 min. Applicare 50μl di acqua deionizzata e ripetere la centrifugazione dei campioni. Questo lavaggio viene ripetuto altre 2 volte, mantenendo la flow-through. </li><li> Se si sta analizzando glicoproteine ​​totale, applicare il sovranatante della lisati (dal punto 9) per il filtro microcontrollore. Lavare l&#39;estratto con 100 l di tampone E, 3 volte per centrifugazione ripetuto a 14000Xg per 3 min. Aggiungere 3 ml di buffer di reazione 10X endo H e 1,5 l di enzima endo H al retentato microcontrollore, e incubare per 3 ore a 37 ° C. I glicani rilasciati vengono eluiti con acqua deionizzata da 3 centrifugazioni ripetute, come al punto 16. </li><li> Se lo si desidera, retentates da passaggi da 16 e 17, contenenti endo H-resistenti glicoproteine ​​vengono poi incubate con 200mU di N-glicosidasi F in 15 ml di tampone C, a 37 ° C per 16 h. Eluizione con acqua deionizzata è fatto come nel passaggio 16. </li><li> Collocare la provetta contenente la reazione endo H flow-through (passaggi da 16 e 17) in un concentratore SpeedVacE asciugare completamente i campioni (questo può essere fatto fino a 45 ° C per accelerare questo processo). </li><li> Per preparare i campioni per HPLC, risospendere il pellet asciutto in 12 ml di solvente per HPLC (acetonitrile: acqua, 60:40, acido fosforico 1%). </li><li> Regolare il dispositivo di HPLC (collegato alla colonna Spherisorb) a flusso costante del solvente (1 ml / min) e pressione (un particolare valore tra 1000 e 2000 psi), e inserire un raccoglitore di frazioni di cambiare un tubo ogni 1 minuto. </li><li> Caricare i campioni nel dispositivo HPLC e avviare il raccoglitore di frazioni contemporaneamente. </li><li> Raccolgono 48 frazioni di 1 ml di ogni campione e correre da una corsa di una miscela standard di oligosaccaridi. </li><li> Trasferire 500 microlitri di ogni frazione di una fiala di scintillazione, e mescolare il contenuto con 3 ml di acqua-liquido miscibile scintillazione. </li><li> Caricare le fiale e leggere in un contatore a scintillazione. </li><li> La lettura cpm viene tracciata in funzione del numero di frazione. </li><li> I valori di cpm all&#39;interno di un picco di rappresentare una determinata specie glycan come segue: frazioni 18-21 corrispondono a M5, M6 frazioni di 22-26, 27-31 frazioni di M7, M8 per 32-35 frazioni, frazioni 36-39 a M9, frazioni 40-42 a 43-45 G1M9 e le frazioni di G2M9. </li><li> La somma dei valori di cpm per le frazioni all&#39;interno di ogni picco riflette la quantità assoluta di una specie glicano specifico. Al fine di compensare il fatto che le specie che contengono più residui di mannosio acquisire più etichetta, la &quot;quantità relativa molare&quot; di ogni specie glicano è calcolato come segue: la quantità assoluta di ciascuna specie glicano è diviso per il numero di residui di mannosio che contiene . Questo valore viene quindi diviso per la somma degli importi relativi molare di tutte le specie glicano ottenute per ottenere il &quot;per cento del totale&quot; per ciascuna specie glycan specifico. </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> Rilasciato con endo H (cpm) un </td><td> 90000 </td><td> 16800 </td></tr><tr><td> Rilasciato con N-glicosidasi F (cpm) b </td><td> 20400 </td><td> 3900 </td></tr><tr><td> Glicoproteine ​​in retentato (cpm) c </td><td> 371700 </td><td> 127695 </td></tr><tr><td> Dolichol-oligosaccaridi a retentato (cpm) d </td><td> 4350 </td><td> 540 </td></tr><tr><td> Dolichol-oligosaccaridi a retentato (%) e </td><td> 1,4 ± 0,7 </td><td> 0,2 ± 0,2 </td></tr></tbody></table> Tabella 1. Analisi del rilascio di N-linked catene di zuccheri con deglycosidases e della presenza di dolichol-oligosaccaridi in campioni glicoproteina. Abbiamo applicato la procedura di pulse-chase ad un lisato di cellule NIH 3T3. Dopo filtrazione microcontrollore, etichettato oligosaccaridi N-linked sono stati rilasciati principalmente con endo H dopo impulso etichettatura (alta tipo di mannosio) e con un ulteriore trattamento con N-glicosidasi F dopo un periodo di caccia, il che corrisponderebbe a tipo complesso (Golgi-lavorati glicani), endo H oligosaccaridi resistenti. Questa analisi quantitativa ha determinato che dolichol-oligosaccaridi, rappresentano una frazione insignificante del marchio nel retentato iniziale. Note: <ol class="lalpha"><li> Cellule NIH 3T3 sono stati etichettati con impulsi [2 - 3 H] mannosio e inseguito con terreno completo. Dopo la lisi cellulare e filtrazione attraverso microcontrollore YM30, alta mannosio oligosaccaridi N-linked sono stati rilasciati da retentates da incubazione con endo H. </li><li> Endo H-materiale resistente in retentates microcontrollore da (a) è stato poi incubato con N-glicosidasi F e rilasciato oligosaccaridi misurata in un contatore a scintillazione. </li><li> Retentates microcontrollore ottenuto come in (a) prima del trattamento endo H, sono stati estratti 4 volte con cloroformio: metanolo: acqua 10:10:03 rimuovere glicolipidi precursore, e il materiale non estratto solubilizzate in NaOH 1N e contate in un contatore a scintillazione. </li><li> Dolichol-oligosaccaridi sono state purificate da estratti (c), come descritto al punto 1 e contate in un contatore a scintillazione. </li><li> Media di 3 esperimenti per cento dei dolichol-oligosaccaridi (d) del cpm totale retentato (c + d). </li></ol> Rappresentante Risultati <img src="/files/ftp_upload/1899/1899fig1.jpg" alt="Figura 1" /> Figura 1. Pulse-chase analisi del totale delle glicoproteine ​​NIH 3T3 in cellule non trattate e sulla inibizione del proteasoma Dopo 1h impulsi etichettatura con. [2 - 3 H] abbiamo ottenuto un profilo atteso con una certa glucosilatiprecursori rimanenti (Glc 2 Man 9 GlcNAc 2 (G2M9) e G1M9), ma la maggior parte delle etichette presenti nella M9 e M8 specie, quest&#39;ultima essendo il risultato di taglio di tutte le glucosio e un residuo mannosio (Figura 1 A). Nessun libero precursori mannosio o altri piccoli sono stati rilevati, che attesta la completezza della purificazione. A seguito di un inseguimento piuttosto lungo 8h c&#39;era più ampio taglio di M7-6 e una piccola quantità di M5, ma la specie più importanti hanno continuato ad essere M9 e M8 (Figura 1 B). Se l&#39;inseguimento è stato fatto in presenza di un inibitore del proteasoma (30 mM MG-132), c&#39;era solo un minore accumulo di queste stesse specie rifilato (Figura 1 C). <img src="/files/ftp_upload/1899/1899fig2.jpg" alt="Figura 2" /> Figura 2. Analisi quantitativa delle catene di zuccheri di un substrato ERAD glicoproteine ​​rispetto al totale. (A) In un esperimento simile a quello nella Figura 1, relativa quantità molare di ogni specie oligosaccaridi sono stati calcolati in base al contenuto mannosio. Abbiamo convertito i valori di cpm ottenuti per ogni specie glicani, la percentuale di ciascuna specie rispetto alla somma totale degli importi relativi molare di tutte le specie presenti è stata poi rappresentata in funzione del tempo la caccia per una media di due esperimenti. (B) simili a (A), tranne che glicani rilasciati dal substrato ERAD ASGPR H2a sono stati analizzati dopo l&#39;etichettatura di impulsi e la caccia per un massimo di 4 ore, in presenza o assenza di inibitori del proteasoma MG-132. (C) Valori per 4h caccia in presenza di MG-132 da (A) e (B) vengono confrontati per ASGPR H2a e la piscina glicoproteina. (D) Schema di N-linked catena zucchero taglio processi al pronto soccorso. Processi di zucchero taglio che portano alla M6-5 legati alle proteine ​​(R) che sono indirizzate a ERAD rispetto a M9-8 su quelle che escono al Golgi e oltre. I risultati indicano che il processo ERAD è associato con il mannosio taglio di N-glicani cedere specie con 5-6 residui di mannosio rimanenti.

<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>

L&#39;impulso-chase analisi dei glicani in cellule vive con la separazione HPLC fornisce un metodo per studiare la dinamica di oligosaccaridi alterazioni strutturali per tutta la vita di una glicoproteina. È sempre più evidente che tali alterazioni sono coinvolte nella produzione dei segnali per la piegatura ER, controllo qualità e il traffico di 2-5 sistemi. Il metodo può essere applicato non solo per una glicoproteina di interesse specifico, ma anche per analizzare le dinamiche della struttura di glicop...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
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pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

Fissaggio del Glc<sub&gt; 3</sub&gt; Uomo<sub&gt; 9</sub&gt; GlcNAc<sub&gt; 2</subPrecursore&gt; oligosaccaride di polipeptidi nascenti al Pronto Soccorso è una modifica comune per le proteine ​​secretorie. Anche se questa modifica è stato coinvolto in diversi processi biologici, ulteriori aspetti della sua funzione stanno emergendo, con recenti evidenze del suo ruolo nella produzione di segnali per il controllo della glicoproteina della qualità e del traffico. Così, fenomeni collegati alla N-glicani legati e la loro elaborazione si stanno intensamente investigato. Metodi che sono stati recentemente sviluppati per l&#39;analisi proteomica hanno notevolmente migliorato la caratterizzazione di glicoproteina N-linked glicani. Tuttavia, essi non forniscono informazioni sulla dinamica del trattamento catena zucchero coinvolti. Per questo, l&#39;etichettatura e la pulse-chase protocolli di analisi che vengono utilizzati di solito sono complesse e danno rese molto basse. Descriviamo qui un metodo semplice per l&#39;isolamento e l&#39;analisi di metabolicamente etichettati oligosaccaridi N-linked. Il protocollo si basa sulla etichettatura delle cellule con [2 -<sup&gt; 3</sup&gt; H] mannosio, lisi enzimatica denaturazione e il rilascio del oligosaccaridi sia da una proteina specifica immunoprecipitato di interesse o dalla piscina glicoproteina generale di trattamenti sequenziali con endo H e N-glicosidasi F, seguita da filtrazione molecolare (Amicon). In questo metodo il oligosaccaridi isolati servire da input per l&#39;analisi HPLC, che consente la discriminazione tra le varie strutture glicani in base al numero di unità di monosaccaride che ne fanno parte, con una risoluzione di un monosaccaride singolo. Usando questo metodo siamo stati in grado di studiare alto mannosio profili oligosaccaridi N-linked di glicoproteine ​​cellulare totale dopo pulse-chase in condizioni normali e sotto l&#39;inibizione del proteasoma. Questi profili sono stati confrontati con quelli ottenuti da un immunoprecipitati ER-associata degradazione (ERAD) substrato. I nostri risultati suggeriscono che la maggior parte NIH 3T3 glicoproteine ​​cellulari sono relativamente stabili e che la maggior parte di loro oligosaccaridi sono tagliata per l&#39;uomo<sub&gt; 9-8</sub&gt; GlcNAc<sub&gt; 2</sub&gt;. Al contrario, i substrati ERAD instabili sono tagliata per l&#39;uomo<sub&gt; 6-5</sub&gt; GlcNAc<sub&gt; 2</sub&gt; E glicoproteine ​​cuscinetto queste specie si accumulano su di inibizione della degradazione del proteasoma.

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

Descriviamo un metodo per l&#39;analisi dell&#39;alterazione di N-glicani legati attraverso la vita inizi di glicoproteine ​​dopo la loro biosintesi in cellule di mammifero. Ciò si ottiene pulse-chase analisi di metabolicamente glicani etichettati, rilascio enzimatico da glicoproteine ​​e l&#39;esame mediante HPLC.

Pulse-chase Analisi di N-linked catene di zuccheri da glicoproteine ​​in cellule di mammifero

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 Views.  Tel Aviv University. Descriviamo un metodo per l'analisi dell'alterazione di N-glicani legati attraverso la vita inizi di glicoproteine ​​dopo la loro biosintesi in cellule di mammifero. Ciò si ottiene pulse-chase analisi di metabolicamente glicani etichettati, rilascio enzimatico da glicoproteine ​​e l'esame mediante HPLC.

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.

In situ Frazionamento subcellulare di cellule di mammifero aderenti e non aderenti

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

Ricerca

Biologia

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.

Analisi del DNA doppio filamento Break (DSB) di riparazione in cellule di mammifero

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.

EsiRNA MISSIONE per lo screening RNAi in cellule di mammifero

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.

Mutagenesi e Analisi funzionale dei canali ionici Heterologously Espresso in cellule di mammifero

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.

Analisi della cinetica di esportazione mRNA nucleare in cellule di mammifero da Microiniezione

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.

Analisi della posizione ciclo cellulare nelle cellule di mammifero

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.

Analisi di espressione di mammifero Linker-istoni Sottotipi

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.

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

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.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

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.

La misurazione dei livelli Heme sintesi in cellule di mammifero

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 ...

Pulse-chase Analisi di N-linked catene di zuccheri da glicoproteine ​​in cellule di mammifero

Pulse-chase Analisi di N-linked catene di zuccheri da glicoproteine in cellule di mammifero