Name: a yeast 2-hybrid screen in batch to compare protein interactions
Uploaded: 2018-06-06T14:30:00.000+00:00
Duration: 14 min 23 s
Description: Watch this Scientific Journal Video about A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions at JoVE.com

Ringraziamo il personale all'interno l'Istituto di genetica umana per il sequenziamento e preparazione di biblioteca NGS. Ringraziamo Einat Snir per la sua esperienza nella preparazione di frammenti di libreria genomica per la libreria di plasmide Y2H fatta qui. Questo lavoro è stato supportato dal National Institutes of Health: R21 NIH EB021870-01A1 e da NSF Research Project Grant: 1517110.

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	<li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Bruckner, A., Polge, C., Lentze, N., Auerbach, D., Schlattner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+two-hybrid,+a+powerful+tool+for+systems+biology.">Yeast two-hybrid, a powerful tool for systems biology.</a> International Journal of Molecular Sciences. 10 (6), 2763-2788 (2009).</li><li>Vidal, M., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+two-hybrid+assay:+still+finding+connections+after+25+years.">The yeast two-hybrid assay: still finding connections after 25 years.</a> Nature Methods. 11 (12), 1203-1206 (2014).</li><li>Pashkova, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DEEPN+as+an+Approach+for+Batch+Processing+of+Yeast+2-Hybrid+Interactions.">DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions.</a> Cell Reports. 17 (1), 303-315 (2016).</li><li>Rajagopala, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Protein-Protein+Interactome+Networks+Using+Yeast+Two-Hybrid+Screens.">Mapping the Protein-Protein Interactome Networks Using Yeast Two-Hybrid Screens.</a> Advances in Experimental Medicine and Biology. 883, 187-214 (2015).</li><li>Weimann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Y2H-seq+approach+defines+the+human+protein+methyltransferase+interactome.">A Y2H-seq approach defines the human protein methyltransferase interactome.</a> Nature Methods. 10 (4), 339-342 (2013).</li><li>Yachie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pooled-matrix+protein+interaction+screens+using+Barcode+Fusion+Genetics.">Pooled-matrix protein interaction screens using Barcode Fusion Genetics.</a> Molecular Systems Biology. 12 (4), 863 (2016).</li><li>Trigg, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrY2H-seq:+a+massively+multiplexed+assay+for+deep-coverage+interactome+mapping.">CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.</a> Nature Methods. , (2017).</li><li>Estojak, J., Brent, R., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+two-hybrid+affinity+data+with+in+vitro+measurements.">Correlation of two-hybrid affinity data with in vitro measurements.</a> Molecular and Cellular Biology. 15 (10), 5820-5829 (1995).</li><li>Rajagopala, S. V., Hughes, K. T., Uetz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+yeast+two-hybrid+systems+using+the+interactions+of+bacterial+motility+proteins.">Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins.</a> Proteomics. 9 (23), 5296-5302 (2009).</li><li>James, P., Halladay, J., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+libraries+and+a+host+strain+designed+for+highly+efficient+two-hybrid+selection+in+yeast.">Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.</a> Genetics. 144 (4), 1425-1436 (1996).</li><li>Barbas, C. F., Burton, D. R., Scott, J. K., Silverman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+DNA+and+RNA.">Quantitation of DNA and RNA.</a> CSH Protoc. 2007, pdb ip47 (2007).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS-Polyacrylamide+Gel+Electrophoresis+of+Proteins.">SDS-Polyacrylamide Gel Electrophoresis of Proteins.</a> Cold Spring Harbor Protocols. 2006 (4), (2006).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences USA. 76 (9), 4350-4354 (1979).</li><li>Alegria-Schaffer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting+using+chemiluminescent+substrates.">Western blotting using chemiluminescent substrates.</a> Methods in Enzymology. 541, 251-259 (2014).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of Visualized Experiments. (62), (2012).</li><li>Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p21+Cdk-interacting+protein+Cip1+is+a+potent+inhibitor+of+G1+cyclin-dependent+kinases.">The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.</a> Cell. 75 (4), 805-816 (1993).</li></ol>

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Qui forniamo una guida per come eseguire le analisi di Y2H in batch utilizzando metodi ottimizzati. Ci sono pochi passaggi critici nella procedura per garantire che la popolazione di lievito che sarebbe stato posto sotto selezione è rappresentante della libreria di partenza e che basta con la popolazione di lievito partenza viene utilizzato per subire la selezione per limitare la variabilità . D'importanza, questi benchmark sono relativamente facili da realizzare a fianco di adattare i metodi e i materiali per un dosag...

Una comprensione completa dei processi biologici delle cellule si basa sull'individuazione delle reti di interazione della proteina che sono alla base della loro meccanismi molecolari. Un approccio per identificare interazioni della proteina è il dosaggio di lievito 2-ibrido (Y2H), che funziona con l'assemblaggio di un fattore di trascrizione chimerica funzionante una volta due dominii della proteina di interesse associare ad uno altro1. Una tipica schermata di Y2H viene eseguita mediante la creazione di una popolazione di lievito che ospita sia una libreria di plasmidi che codificano le proteine interagenti fuse ad un attivatore trascrizionale (ad es., 'preda' proteina di fusione) e un plasmide dato 'esca' composta la proteina di interesse fusa a un dominio di legame del DNA (per esempio, il dominio legante il DNA di Gal4 che lega la sequenza d'attivazione Gal4-upstream). Uno dei principali vantaggi dell'approccio Y2H è che è relativamente facile e poco costoso per condurre in un tipico laboratorio attrezzato per lavoro biologico molecolare routine2. Tuttavia, quando tradizionalmente eseguita, un utente campioni diverse colonie che sorgono al momento della selezione per una positiva interazione Y2H. Questo limita fortemente il numero di cloni 'preda' libreria che può essere oggetto di indagine. Questo problema è aggravato quando l'abbondanza di una particolare preda interagente è molto alta rispetto alle altre, diminuendo la possibilità di rilevare l'interazione da plasmidi preda abbondanza bassa.
Una soluzione per l'utilizzo del principio di Y2H in una copertura completa del proteoma è l'uso di un approccio di matrice-formattato in cui una matrice contenente noto preda singoli plasmidi possa essere interrogata digitalmente. Tuttavia, tale approccio richiede un'infrastruttura che non è facilmente accessibile e conveniente per diversi ricercatori che sono interessati nella definizione Interactoma di un piccolo numero di proteine o domini3. Inoltre, librerie di prede molto complessa che possono codificare più frammenti di proteine interagenti sarebbero espandere le dimensioni di tali matrici matrice ai formati impraticabile. Un'alternativa consiste nell'eseguire saggi con librerie complesse in lotti e valutare la presenza di cloni interagenti mediante sequenziamento massivo di high throughput parallelo4. Questo può essere applicato per analizzare la presenza di prede plasmidi che sorgono nelle colonie multiple utilizzando un tipico Y2H formattato approccio nel quale lievito cellule alloggiamento un interagenti delle proteine di fusione sono autorizzate a crescere su una piastra5,6. Questa idea generale può essere accentuata per aumentare query di entrambi più esche e prede componenti al stesso tempo7,8.
Ancora, molte indagini richiedono che un più facile ancora più concentrati sforzo sulla proteina a pochi 'esche' e possono beneficiare più di una query esaustiva e semi-quantitativa di una libreria singola preda complessi. Abbiamo sviluppato e convalidato un metodo per eseguire gli studi di interazione della proteina su vasta scala utilizzando un principio di Y2H in batch formato4. Questo utilizza il tasso di espansione di un plasmide particolare preda come un proxy per la forza relativa di Y2H interazione9. Sequenziamento profondo di tutti i plasmidi all'interno di una popolazione sottoposta alla normale crescita o condizioni di crescita selettiva produce una mappa completa dei cloni che producono interazioni Y2H forti e deboli. Il repertorio di interattori possa essere ottenuto e confrontato direttamente attraverso più esca plasmidi. Il flusso di lavoro risultante definito profondo (arricchimento dinamico per la valutazione della proteina reti) quindi utilizzabile per identificare differenziale Interattomi dalle stesse librerie di preda per identificare le proteine, permettendo un confronto tra una proteina contro un altro.
Qui, noi dimostrare profondo e introdurre miglioramenti nei metodi di laboratorio che facilitano l'uso, che sono illustrati nella Figura 1. Miglioramenti significativi includono:
Generazione di popolazioni lievito preda. Uno dei requisiti chiavi di profondo sta generando popolazioni di lievito con i plasmidi di esche diverse che hanno la stessa distribuzione delle librerie preda del plasmide. Popolazioni di equivalente della linea di base della libreria plasmide preda sono essenziali per effettuare confronti accurati tra l'interattomi di diverse esche. Ciò si ottiene quando un plasmide di biblioteca occupa già una popolazione di lievito aploidi e lo spostamento di un plasmide dato esca in quella popolazione è ottenuto dall'accoppiamento per produrre un diploide. Qui, forniamo una guida chiara su come rendere tali popolazioni utilizzando librerie commerciali ospitate in lievito aploide. Anche se abbiamo trovato metodi che generano un elevato numero di diploidi, l'efficienza complessiva degli amori di questi ceppi di lievito commerciale libreria-contenente era basso. Di conseguenza, abbiamo costruito un nuovo ceppo che può ospitare la preda librerie che produce molto più diploidi per reazione di accoppiamento.
Nuovo set di plasmidi di esca. Molti plasmidi corrente che esprimono proteine di fusione 'esca' comprende della proteina di interesse e un dominio di legame al DNA sono basati su 2 µ, permettendo loro di amplificare il loro numero di copia. Questo numero di copia può essere abbastanza variabile nella popolazione e portare alla variabilità della risposta trascrizionale di Y2H. Questo a sua volta potrebbe inclinare la capacità di misurare la forza di un'interazione proteina data sulla base della risposta di crescita delle cellule in selezione. Questo può essere parzialmente indirizzo utilizzando un plasmide copia basso, alcuni dei quali precedentemente sono stati descritti come il pDEST32 commercialmente disponibili10. Abbiamo costruito un nuovo plasmide esca (pTEF-GBD) che produce proteine di fusione di dominio Gal4-DNA-binding all'interno di un plasmide copia basso basato su centromero TRP1 che trasporta il gene di resistenza Kanr che permette anche la clonazione di frammenti di esca sia a Monte e a valle del dominio legante il DNA di Gal4.
Libreria di frammento di nuovo ad alta densità Y2H. Abbiamo costruito un nuovo plasmide per librerie di casa Y2H preda e usato per costruire una libreria di Y2H altamente complessa composta casualmente tranciati frammenti di DNA genomico da Saccharomyces cerevisiae. Analisi di sequenza ha indicato che questa libreria ha avuto oltre 1 milione di elementi diversi, molto più complessi di lievito precedentemente descritti genomica Y2H plasmide biblioteche11. Con questa nuova libreria, siamo stati in grado di dimostrare che il flusso di lavoro profondo è abbastanza robusto per ospitare librerie complesse con molti plasmidi differenti in modo che sia affidabile e riproducibile.

<table><tbody><tr><td>Illumina HiSeq 4000</td><td>Illumina</td><td></td><td>deep sequencing platform</td></tr><tr><td>Monoclonal anti-HA antibodies</td><td>Biolegend</td><td>901514</td><td>Primary Antibody to detect expression of HA in pGal4AD constructs</td></tr><tr><td>Polyclonal anti-myc antibodies</td><td>QED Biosciences Inc</td><td>18826</td><td>Primary Antibody to detect expression of MYC in pTEF-GBD constructs</td></tr><tr><td>NarI</td><td>New England BioLabs</td><td>R0191S</td><td></td></tr><tr><td>EcoRI-HF</td><td>New England BioLabs</td><td>R3101S</td><td></td></tr><tr><td>BamHI-HF</td><td>New England BioLabs</td><td>R3236S</td><td></td></tr><tr><td>XhoI</td><td>New England BioLabs</td><td>R0146S</td><td></td></tr><tr><td>Polyethylene Glycol 3350, powder</td><td>J.T. Baker</td><td>U2211-08</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Trevigen, Inc sold by Fisher Scientific</td><td>50-948-286</td><td>carrier DNA for yeast transformation section 3.2.1.</td></tr><tr><td>Kanamycin Monosulfate</td><td>Research Products International</td><td>K22000</td><td></td></tr><tr><td>LE Agarose</td><td>GeneMate</td><td>E-3120-500</td><td>used for making DNA agarose gels</td></tr><tr><td>Sodium Chloride</td><td>Research Products International</td><td>S23025</td><td></td></tr><tr><td>Tryptone</td><td>Research Products International</td><td>T60060</td><td></td></tr><tr><td>D-Sorbitol</td><td>Research Products International</td><td>S23080</td><td></td></tr><tr><td>Lithium Acetate Dihydrate</td><td>MP Biomedicals</td><td>155256</td><td></td></tr><tr><td>Calcium Chloride</td><td>ThermoFisher</td><td>C79</td><td></td></tr><tr><td>EDTA Sodium Salt</td><td>Research Products International</td><td>E57020</td><td></td></tr><tr><td>Yeast Extract Powder</td><td>Research Products International</td><td>Y20020</td><td></td></tr><tr><td>Yeast Nitrogen Base (ammonium sulfate) w/o amino acids</td><td>Research Products International</td><td>Y20040</td><td></td></tr><tr><td>CSM-Trp-Leu+40ADE</td><td>Formedium</td><td>DCS0789</td><td></td></tr><tr><td>CSM-Trp-Leu-His+40ADE</td><td>Formedium</td><td>DCS1169</td><td></td></tr><tr><td>CSM-Leu-Met</td><td>Formedium</td><td>DCS0549</td><td></td></tr><tr><td>CSM-Trp-Met</td><td>Bio 101, Inc</td><td>4520-922</td><td></td></tr><tr><td>L-Methionine</td><td>Formedium</td><td>DOC0168</td><td></td></tr><tr><td>Adenine</td><td>Research Products International</td><td>A11500</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Research Products International</td><td>G32045</td><td></td></tr><tr><td>Bacto Agar</td><td>BD</td><td>214010</td><td>used for making media plates in section 1</td></tr><tr><td>Peptone</td><td>Research Products International</td><td>P20240</td><td></td></tr><tr><td>3-amino-1,2,4 Triazole</td><td>Sigma</td><td>A8056</td><td></td></tr><tr><td>2-Mercaptoehanol (BME)</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>Zymolyase 100T</td><td>USBiological</td><td>Z1004</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma</td><td>P8281</td><td></td></tr><tr><td>Phenol:Chloroform:IAA</td><td>Ambion</td><td>AM9732</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma-Aldrich</td><td>238074</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories, INC</td><td>2716</td><td></td></tr><tr><td>RNAse A</td><td>ThermoFisher</td><td>EN0531</td><td></td></tr><tr><td>Urea</td><td>Research Products International</td><td>U20200</td><td></td></tr><tr><td>SDS</td><td>Research Products International</td><td>L22010</td><td></td></tr><tr><td>glycerol</td><td>Sigma Aldrich</td><td>G5516</td><td></td></tr><tr><td>Tris-HCl</td><td>Gibco</td><td>15506-017</td><td></td></tr><tr><td>bromophenol blue</td><td>Amresco</td><td>449</td><td></td></tr><tr><td>Gibson Assembly Cloning Kit</td><td>New England Biolabs</td><td>E5510S</td><td>Rapid assembly method for cloning of plasmids in section 2</td></tr><tr><td>NEBNext High-Fidelity 2x PCR Master Mix</td><td>New England Biolabs</td><td>M0541S</td><td>Used for amplification of products for Gibson Assembly in Section 2.3 as well assample preparation for DEEPN deep sequencing in section 6.2.1</td></tr><tr><td>Ethidium Bromide</td><td>Amresco</td><td>0492-5G</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>28104</td><td>Used for purification of pcr products in section 6.2.3</td></tr><tr><td>Qiaquick DNA Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>Used for purification of digested pTEF-GBD in section 2.1</td></tr><tr><td>KAPA Hyper Prep kit</td><td>KAPA Biosystems</td><td>KK8500</td><td>preparation kit for deep sequencing</td></tr><tr><td>Codon optimization</td><td>http://www.jcat.de</td><td></td><td></td></tr><tr><td>Codon optimization</td><td>https://www.idtdna.com/CodonOpt</td><td></td><td></td></tr><tr><td>gBlocks</td><td>Integrated DNA Technologies</td><td></td><td>DNA fragments used for cloning in Section 2.2</td></tr><tr><td>Strings</td><td>Thermofisher</td><td></td><td>DNA fragments used for cloning in Section 2.2</td></tr><tr><td>GenCatch Plasmid DNA mini-prep Kit</td><td>EPOCH Life Sciences</td><td></td><td>Used to prepare quantities of DNA in Section 2.3</td></tr><tr><td>Covaris E220</td><td>Covaris</td><td></td><td>high performance ultra-sonicator in section 7</td></tr><tr><td>oligo nucelotide 5&amp;rsquo;- CGGTCTT&lt;br/&gt; CAATTTCTCAAGTTTCAG -3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>used for pcr amplification and sequencing 5&#039; insert pTEF-GBD during plasmid construction</td></tr><tr><td>oligo nucelotide 5&amp;rsquo;-GAGTAACG&lt;br/&gt; ACATTCCCAGTTGTTC-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>used for pcr amplification and sequencing 5&#039; insert pTEF-GBD during plasmid construction</td></tr><tr><td>oligo nucelotide 5&amp;rsquo;-CACCGTAT&lt;br/&gt; TTCTGCCACCTCTTCC-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>used for pcr amplification and sequencing 3&#039; insert pTEF-GBD during plasmid construction</td></tr><tr><td>oligo nucelotide 5&amp;rsquo;-GCAACCGC&lt;br/&gt; ACTATTTGGAGCGCTG-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>used for pcr amplification and sequencing 3&#039; insert pTEF-GBD during plasmid construction</td></tr><tr><td>oligonucleotide 5&amp;rsquo;-GTTCCGATG&lt;br/&gt; CCTCTGCGAGTG-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>5&#039; Pimer used for insert amplification of pGAL4AD</td></tr><tr><td>oligonucelotide 5&amp;rsquo;-GCACATGCT&lt;br/&gt; AGCGTCAAATACC-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>3&#039; Pimer used for insert amplification of pGAL4AD</td></tr><tr><td>oligonucelotide 5&amp;rsquo;-ACCCAAGCA&lt;br/&gt; GTGGTATCAACG-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>5&#039; Pimer used for insert amplification of pGADT7</td></tr><tr><td>oligonucelotide 5&amp;rsquo;- TATTTAGA&lt;br/&gt; AGTGTCAACAACGTA -3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>3&#039; Pimer used for insert amplification of pGADT7</td></tr><tr><td>PJ69-4A MatA yeast strain</td><td>http://depts.washington.edu/yeastrc/ James P, Halladay J, Craig EA: Genomic Libraries and a host strain designed for highly efficient two-hybrid selection in yeast. GENETICS 1996 144:1425-1436</td><td></td><td>MATA leu2-3,112 ura3-52 trp1-901 his3-200 gal4D, gal80D, GAL-ADE2 lys2::GAL1-HIS3 met2::GAL7</td></tr><tr><td>pTEF-GBD</td><td>Dr. Robert Piper Lab</td><td></td><td>Gal4-DNA binding doimain expression plasmid</td></tr><tr><td>pGal4AD (pPL6343)</td><td>Dr. Robert Piper Lab</td><td></td><td>Gal4-activation domain expression plasmid</td></tr><tr><td>100 mm petri dishes</td><td>Kord-Vallmark sold by VWR</td><td>2900</td><td></td></tr><tr><td>125 mL PYREX Erlenmeyer flask</td><td>Fisher Scientific</td><td>S63270</td><td></td></tr><tr><td>250 mL PYREX Erlenmeyer flask</td><td>Fisher Scientific</td><td>S63271</td><td></td></tr><tr><td>1,000 mL PYREX Erlenmeyer flask</td><td>Fisher Scientific</td><td>S63274</td><td></td></tr><tr><td>2,000 mL PYREX Erlenmeyer flask</td><td>Fisher Scientific</td><td>S63275</td><td></td></tr><tr><td>20 X 150 mm Disposable Culture Tube</td><td>Thermofisher</td><td>14-961-33</td><td></td></tr><tr><td>pipet-aid</td><td>Drummond</td><td>4-000-100</td><td></td></tr><tr><td>5 mL Serological Pipette</td><td>Denville</td><td>P7127</td><td></td></tr><tr><td>10 mL Serological Pipette</td><td>Denville</td><td>P7128</td><td></td></tr><tr><td>25 mL Serological Pipette</td><td>Denville</td><td>P7129</td><td></td></tr><tr><td>1,000 mL PYREX Griffin Beaker</td><td>Fisher Scientific</td><td>02-540P</td><td></td></tr><tr><td>1,000 mL PYREX Reuasable Media Storage Bottle</td><td>Fisher Scientific</td><td>06-414-1D</td><td></td></tr><tr><td>1,000 mL graduated cylinder</td><td>Fisher Scientific</td><td>08-572-6G</td><td></td></tr><tr><td>SpectraMax 190</td><td>Molecular Devices</td><td></td><td>used to measure the Optical Density of cells</td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>ND-2000</td><td>Spectrophotometer used to quantify amount of DNA</td></tr><tr><td>Electronic UV transilluminator</td><td>Ultra Lum</td><td>MEB 20</td><td>used to visualize DNA in an Ethidium Bromide agarose gel</td></tr><tr><td>P1000 Gilson PIPETMAN</td><td>Fisher Scientific</td><td>F123602G</td><td></td></tr><tr><td>P200 Gilson PIPETMAN</td><td>Fisher Scientific</td><td>F123601G</td><td></td></tr><tr><td>P20 Gilson PIPETMAN</td><td>Fisher Scientific</td><td>F123600G</td><td></td></tr><tr><td>P10 Gilson PIPETMAN</td><td>Fisher Scientific</td><td>F144802G</td><td></td></tr><tr><td>1250 &amp;micro;L Low Retention Pipette Tips</td><td>GeneMate</td><td>P-1236-1250</td><td></td></tr><tr><td>200 &amp;micro;LLow Retention Pipette Tips</td><td>VWR</td><td>10017-044</td><td></td></tr><tr><td>10 &amp;micro;L XL Low Retention Pipette Tips</td><td>VWR</td><td>10017-042</td><td></td></tr><tr><td>50 mL conical tube</td><td>VWR</td><td>490001-627</td><td></td></tr><tr><td>15 mL conical tube</td><td>VWR</td><td>490001-621</td><td></td></tr><tr><td>cell scraper</td><td>Denville Scientific</td><td>TC9310</td><td></td></tr><tr><td>1.5 mL Microcentrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>HCl</td><td>Fluka Analytical</td><td>318949-1L</td><td></td></tr><tr><td>NaOH</td><td>J.T. Baker</td><td>5674-02</td><td></td></tr><tr><td>Wooden applicators</td><td>Solon Care</td><td>55900</td><td></td></tr><tr><td>Eppendorf microcentrifuge 5424</td><td>Fisher Scientific</td><td>05-400-005</td><td>microcentrifuge</td></tr><tr><td>Sorvall ST16R</td><td>Thermo Fisher Scientific</td><td>75004381</td><td>benchtop centrifuge</td></tr><tr><td>Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey)</td><td>GE Healthcare</td><td>NA934-1ML</td><td>Secondary Antibody</td></tr><tr><td>Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep)</td><td>GE Healthcare</td><td>NA931-1ML</td><td>Secondary Antibody</td></tr><tr><td>SuperSignal West Pico Chemiluminescent Substrate</td><td>Thermo Fisher Scientific</td><td>34080</td><td>ECL detection solution</td></tr><tr><td>Isotemp Incubator</td><td>Thermo Fisher Scientific</td><td></td><td>Incubator</td></tr><tr><td>Mutitron 2</td><td>INFORS HT</td><td></td><td>Shaking incubator</td></tr><tr><td>Isotemp Digital-Control Water Bath Model 205</td><td>Fisher Scientific</td><td></td><td>water bath</td></tr><tr><td>Y2H mouse cDNA library in Y187 (pan tissue)</td><td>Clontech</td><td>630482</td><td>commercially available cDNA Library</td></tr><tr><td>Y2H mouse cDNA library in Y187 (mouse brain)</td><td>Clontech</td><td>630488</td><td>commercially available cDNA Library</td></tr><tr><td>pGADT7 AD Vector</td><td>Clontech</td><td>630442</td><td>commercially available AD Vector housing many cDNA libraries</td></tr><tr><td>pGBKT7 DNA-BD Vector</td><td>Clontech</td><td>630443</td><td>commercially available DNA-BD Vector</td></tr><tr><td>Biolase DNA Polymerase</td><td>Bioline</td><td>BIO-21042</td><td>DNA polymerase used for section 2.4</td></tr><tr><td>GeneMate GCL-60 Thermal Cycler</td><td>BioExpress</td><td>P-6050-60</td><td>pcr machine</td></tr><tr><td>TempAssure 0.5 mL PCR tubes</td><td>USA Scientific</td><td>1405-8100</td><td></td></tr></tbody></table>

a yeast 2-hybrid screen in batch to compare protein interactions

Lo screening per le interazioni proteina-proteina usando il dosaggio di 2-ibrido del lievito è stato a lungo uno strumento efficace, ma il suo utilizzo in gran parte è stato limitato alla scoperta di interattori di alto-affinità che sono altamente arricchito nella biblioteca di candidati interagenti. In un formato tradizionale, il dosaggio di 2-ibrido del lievito può produrre troppo molte colonie per analizzare quando condotto presso stringenza dove potrebbero essere trovati Interactiani bassa affinità. Inoltre, senza un interrogatorio esaustivo e completo della stessa libreria contro esca diversi plasmidi, un'analisi comparativa non possono essere realizzata. Anche se alcuni di questi problemi possono essere risolto utilizzando librerie allinearono preda, il costo e l'infrastruttura necessaria per far funzionare questi schermi possono essere proibitivi. In alternativa, abbiamo adattato il dosaggio di 2-ibrido del lievito per scoprire contemporaneamente decine di transitori e interazioni proteina statico all'interno di un unico schermo che utilizzano una strategia definita profondo (arricchimento dinamico per la valutazione della proteina reti), che incorpora il sequenziamento del DNA di alto-rendimento e calcolo per seguire l'evoluzione di una popolazione di plasmidi che codificano interattori. Qui, descriviamo su misura reagenti e protocolli che consentono un profondo schermo per essere eseguito facilmente e a costi contenuti.

Il dosaggio di Y2H è stato ampiamente utilizzato per la ricerca di interazioni proteina/proteina e diversi adattamenti e sistemi sono stati sviluppati. Per la maggior parte, le stesse considerazioni che aiutano a garantire il successo con questi approcci precedenti sono importanti per profondo. Sono alcuni dei punti di riferimento importanti: garantire espressione del dominio di legame al DNA proteine di fusione, garantendo un basso fondo di spurie suo + crescita in diploidi contenente l...

Watch this Scientific Journal Video about A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions at JoVE.com

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Una schermata di 2-ibrido del lievito in Batch per confrontare interazioni proteina

Elaborazione batch delle schermate di lievito 2-ibrido consente di confronto diretto tra i profili di interazione delle proteine multiple esca con un insieme molto complesso di proteine di fusione di preda. Qui, descriviamo raffinati metodi, nuovi reagenti e come implementare il loro utilizzo per tali schermi.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

a-yeast-2-hybrid-screen-in-batch-to-compare-protein-interactions

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

13.3K Views.  University of Iowa. Elaborazione batch delle schermate di lievito 2-ibrido consente di confronto diretto tra i profili di interazione delle proteine multiple esca con un insieme molto complesso di proteine di fusione di preda. Qui, descriviamo raffinati metodi, nuovi reagenti e come implementare il loro utilizzo per tali schermi.

Video: A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

Un lievito modificato - un sistema ibrido per studi di interazione tra DNA e proteine ​​eteromeriche

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Ricerca

Biochimica

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

Informatica analisi dei dati di sequenza da Batch lievito ibrido 2 schermi

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

Genetica

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Un approccio comparato alla caratterizzano il paesaggio di ospite-patogeno interazioni proteina-proteina

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Immunologia e infezioni

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitina membrana a base di lievito Due-Hybrid (MITO) Sistema: un potente strumento per identificare interazioni proteina-proteina

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Biologia

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Svelare la funzione di un batterica Effector da un non-coltivabili fitopatogeni Utilizzando un lievito schermo doppio ibrido

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificato lievito-sistema del doppio ibrido per identificare proteine ​​che interagiscono con il fattore di crescita progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

Probing ad alta densità di microarrays della proteina funzionali per rilevare le interazioni proteina-proteina

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Approcci genetici e biochimici per<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; La valutazione di proteine ​​Oligomerizzazione: Il rianodina Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Studi di interazione biomolecolare utilizzando la tecnica dei due ibridi di lievito 

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Autore in primo piano: Isolamento migliorato di mutanti nulli di interazione nel lievito

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Isolamento di mutanti nulli/alterati di interazione utilizzando il saggio a due ibridi di lievito

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

Una schermata di 2-ibrido del lievito in Batch per confrontare interazioni proteina

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un lievito modificato - un sistema ibrido per studi di interazione tra DNA e proteine eteromeriche

Informatica analisi dei dati di sequenza da Batch lievito ibrido 2 schermi

Un approccio comparato alla caratterizzano il paesaggio di ospite-patogeno interazioni proteina-proteina

Split-Ubiquitina membrana a base di lievito Due-Hybrid (MITO) Sistema: un potente strumento per identificare interazioni proteina-proteina

Svelare la funzione di un batterica Effector da un non-coltivabili fitopatogeni Utilizzando un lievito schermo doppio ibrido

Modificato lievito-sistema del doppio ibrido per identificare proteine che interagiscono con il fattore di crescita progranulina

Probing ad alta densità di microarrays della proteina funzionali per rilevare le interazioni proteina-proteina

Approcci genetici e biochimici per

Isolamento di mutanti nulli/alterati di interazione utilizzando il saggio a due ibridi di lievito

Isolamento di mutanti nulli/alterati di interazione utilizzando il saggio a due ibridi di lievito

Una schermata di 2-ibrido del lievito in Batch per confrontare interazioni proteina

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un lievito modificato - un sistema ibrido per studi di interazione tra DNA e proteine ​​eteromeriche

Informatica analisi dei dati di sequenza da Batch lievito ibrido 2 schermi

Un approccio comparato alla caratterizzano il paesaggio di ospite-patogeno interazioni proteina-proteina

Split-Ubiquitina membrana a base di lievito Due-Hybrid (MITO) Sistema: un potente strumento per identificare interazioni proteina-proteina

Svelare la funzione di un batterica Effector da un non-coltivabili fitopatogeni Utilizzando un lievito schermo doppio ibrido

Modificato lievito-sistema del doppio ibrido per identificare proteine ​​che interagiscono con il fattore di crescita progranulina

Probing ad alta densità di microarrays della proteina funzionali per rilevare le interazioni proteina-proteina

Approcci genetici e biochimici per

Isolamento di mutanti nulli/alterati di interazione utilizzando il saggio a due ibridi di lievito

Isolamento di mutanti nulli/alterati di interazione utilizzando il saggio a due ibridi di lievito

Un lievito modificato - un sistema ibrido per studi di interazione tra DNA e proteine eteromeriche

Modificato lievito-sistema del doppio ibrido per identificare proteine che interagiscono con il fattore di crescita progranulina