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

Wir danken dem Mitarbeiter im Institut für Humangenetik für NGS Bibliothek Vorbereitung und Sequenzierung. Wir danken für ihre Expertise bei der Vorbereitung genomische Bibliothek Fragmente für die Y2H-Plasmid-Bibliothek hier Einat Snir. Diese Arbeit wurde vom National Institutes of Health unterstützt: NIH R21 EB021870-01A1 und durch 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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Hier bieten wir Ihnen eine Anleitung für Y2H-Assays im Batch mit optimierten Methoden durchführen. Es gibt ein paar wichtige Schritte im Verfahren um sicherzustellen, dass die Bevölkerung von Hefe, die Auswahl unterstellt würde Vertreter der Start-Bibliothek ist, genug der Ausgangspunkt Hefe Bevölkerung dient zur Auswahl um Variabilität zu begrenzen zu unterziehen . Diese Benchmarks sind relativ leicht zu erreichen, neben der Anpassung der Methoden und Materialien für einen traditionellen Y2H-Assay, wodurch dieser...

Ein vollständiges Verständnis der biologischen Zellprozesse stützt sich auf die Suche nach der Protein-Interaktion-Netzwerke, die ihre molekularen Mechanismen zugrunde liegen. Ein Ansatz zur Protein-Interaktionen zu identifizieren ist die Hefe 2-Hybrid (Y2H) Assay, die Arbeiten durch einen funktionierenden Chimären Transkriptionsfaktor zusammenfügen, sobald zwei Protein Domänen von Interesse an einander1binden. Ein typische Y2H-Bildschirm erfolgt durch die Schaffung einer Bevölkerung von Hefe, die beide eine Bibliothek von Plasmiden Codierung interagierenden Proteine verschmolzen zu einem transcriptional Aktivator beherbergt (zB., "Beute" Schmelzverfahren Protein) und ein bestimmtes "Köder" Plasmid bestehend aus dem Protein von Interesse verschmolzen zu einem DNA-bindende Domäne (z.B. die Gal4 DNA-bindende Domäne, die an die Gal4-upstream aktivierende Sequenz bindet). Einer der Hauptvorteile des Y2H-Ansatzes ist, dass es relativ einfach ist und kostengünstig durchzuführen in einem typischen Labor für Routinearbeiten molekularen biologischen2 ausgestattet. Jedoch wenn traditionell durchgeführt, Proben ein Benutzer einzelne Kolonien, die bei der Auswahl für eine positive Y2H-Interaktion entstehen. Dies schränkt die Anzahl der Bibliothek "Beute"-Klone, die befragt werden können. Dieses Problem wird verstärkt, wenn die Fülle an eine bestimmte interagierenden Beute sehr hoch im Vergleich zu anderen, verringern die Chance Interaktion von niedrigen Fülle Beute Plasmide zu erkennen ist.
Eine Lösung für die Verwendung der Y2H-Prinzip in umfassende Abdeckung des Proteoms ist die Verwendung eines Matrix-formatierte Ansatzes, wobei ein Array mit bekannten individuelle Beute Plasmide Digital abgefragt werden kann. Ein solches Vorgehen erfordert jedoch eine Infrastruktur, die nicht ohne weiteres zugänglich oder kostengünstig an die einzelnen Ermittler ist, die bei der Festlegung der Interaktom einer kleinen Anzahl von Proteinen oder Domänen3interessiert sind. Darüber hinaus würde sehr komplexe Beute-Bibliotheken, die mehrere Fragmente von interagierenden Proteine kodieren können solche Matrix Arrays zu unpraktisch Größen vergrößern. Eine Alternative ist Assays mit komplexen Bibliotheken in Chargen und das Vorhandensein von interagierenden Klone mit massiv parallelen Hochdurchsatz-Sequenzierung4zu bewerten. Dies kann angewendet werden, um das Vorhandensein von Beute Plasmide assay, die in mehrere Kolonien mit einem typischen entstehen Y2H formatiert Ansatz in der Hefe Zellen Gehäuse interagierenden Pair von Fusionsproteinen dürfen auf einer Platte5,6wachsen. Diese allgemeine Idee kann akzentuiert werden, zur Steigerung der Abfrage der beiden mehrere Köder und Komponenten in der gleichen Zeit7,8zum Opfer.
Dennoch, viele Untersuchungen erfordern, dass ein einfacher noch mehr Aufwand auf nur wenige Protein "Köder" konzentriert und profitieren mehr von einer erschöpfenden und semi-quantitativen Abfrage einer einzigen komplexen Beute-Bibliothek. Wir haben entwickelt und validiert ein Konzept für die breit angelegte Protein Wechselwirkungsstudien mit einem Y2H-Prinzip in Batch Format4durchführen. Dies nutzt das Wachstumstempo von einem bestimmten Beute Plasmid als Proxy für die relative Stärke der Y2H Interaktion9. Tiefe Sequenzierung aller Plasmide innerhalb einer Population normales Wachstum ausgesetzt oder selektive Wachstumsbedingungen produziert eine vollständige Karte der Klone, die starke und schwache Y2H Interaktionen ergeben. Das Repertoire der interagierenden werden erhalten und direkt über mehrere Köder Plasmide verglichen. Die resultierende Workflow bezeichnet DEEPN (dynamische Bereicherung für Evaluation of Protein Networks) kann so zum differenziellen Interactomes aus den gleichen Beute Bibliotheken, Proteine, Vergleich zwischen ein Protein vs. anderen zu identifizieren identifizieren.
Hier zeigen wir DEEPN und Verbesserungen in die Labormethoden, die seine Verwendung zu erleichtern, die in Abbildung 1aufgeführt sind. Signifikante Verbesserungen umfassen:
Generation der Beute Hefe Bevölkerung. Eine der wichtigsten Voraussetzungen der DEEPN erzeugt Populationen von Hefe mit verschiedenen Köder Plasmide, die die gleiche Verteilung der Plasmid-Beute-Bibliotheken haben. Gleichwertige Grundlinie Populationen der Beute Plasmid Bibliothek sind essentiell für genaue Vergleiche zwischen den Interactomes von unterschiedlichen Ködern. Dies geschieht am besten, wenn ein Bibliothek-Plasmid sich bereits in einer Population von haploiden Hefe befindet und Umzug eines bestimmten Köder-Plasmid in dieser Population erreicht wird, indem die Paarung um einen diploiden zu produzieren. Hier bieten wir einen klaren Leitfaden wie man solche Populationen mit kommerziellen Bibliotheken in haploiden Hefe untergebracht. Obwohl wir Methoden, die eine hohe Anzahl von diploiden zu erzeugen gefunden, war die Paarung Gesamteffizienz des diese kommerzielle Bibliothek-haltigen Hefestämme gering. Daher haben wir eine neue Sorte, die Beute Bibliotheken aufnehmen kann, die weit mehr diploiden pro Paarung Reaktion ergibt.
New set Köder Plasmide. Viele aktuelle Plasmide, die "Köder" Schmelzverfahren Proteine enthalten des Proteins des Interesses und eine DNA-bindende Domäne Ausdrücken basieren auf 2µ, so dass sie ihre Kopienzahl verstärken. Diese Kopienzahl kann sehr variabel in der Bevölkerung und führen zu Schwankungen in der Y2H transcriptional Antwort. Dies könnte wiederum die Fähigkeit zu beurteilen, die Stärke einer bestimmten Protein Interaktion anhand der Antwort Wachstum von Zellen unter Auswahl verzerren. Dies kann teilweise Adresse sein, mit einem niedrigen Kopie Plasmid, von denen einige wie die im Handel erhältlichen pDEST3210zuvor beschrieben wurden. Wir konstruierten eine neue Köder-Plasmid (pTEF-GBD), das produziert Gal4 DNA-bindende Domäne Fusionsproteine innerhalb einer TRP1 Zentromer-basierte niedrigen Kopie Plasmid trägt die Kanr-Resistenz-Gen, die auch ermöglicht, Klonen von Köder Fragmente sowohl stromaufwärts und unterhalb des Gal4 DNA-bindende Domäne.
Neue High-Density Y2H fragmentbibliothek. Wir ein neues Plasmid, Haus Y2H Beute Bibliotheken gebaut und benutzte es, um eine hochkomplexe Y2H-Bibliothek nach dem Zufallsprinzip geschert Bruchstücke von genomischer DNA aus Saccharomyces Cerevisiaegemacht zu bauen. Sequenzanalyse zeigte, dass diese Bibliothek mehr als 1 Million verschiedene Elemente, die weitaus komplexer als die zuvor beschriebenen Hefe genomische Y2H Plasmid Bibliotheken11. Mit dieser neuen Bibliothek konnten wir zeigen, dass der DEEPN-Workflow robust genug ist, um komplexe Bibliotheken mit vielen unterschiedlichen Plasmide in einer Art und Weise aufzunehmen, die zuverlässig und reproduzierbar ist.

<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

Screening für Protein-Protein-Interaktionen mit der Hefe-2-Hybrid-Assay ist seit langem ein wirksames Instrument, aber seine Verwendung wurde weitgehend auf die Entdeckung der hohen Affinität Interaktoren, die in der Bibliothek der interagierenden Kandidaten hochangereichertes sind begrenzt. In einem traditionellen Format kann Hefe 2-Hybrid Assay zu viele Kolonien zu analysieren, wenn bei niedrigen Stringenz durchgeführt ergeben, wo geringe Affinität Interaktoren gefunden werden könnte. Darüber hinaus kann nicht ohne eine umfassende und vollständige Befragung derselben Bibliothek gegen verschiedene Köder Plasmide, eine vergleichende Analyse erreicht werden. Obwohl einige dieser Probleme behoben werden kann, angeordneten Beute Bibliotheken verwenden, können die Kosten und die erforderliche Infrastruktur für solche Bildschirme betreiben unerschwinglich sein. Als Alternative haben wir die Hefe 2-Hybrid Assay um gleichzeitig entdecken Sie Dutzende von Transienten angepasst und statische Protein-Interaktionen in einem einzigen Bildschirm mit Hilfe einer Strategie bezeichnet DEEPN (dynamische Bereicherung für Evaluation of Protein Networks), die enthält Hochdurchsatz-DNA-Sequenzierung und Berechnung, mit der die Entwicklung einer Population von Plasmiden folgen, die Interaktionspartnern zu kodieren. Hier beschreiben wir maßgeschneiderte Reagenzien und Protokolle, die einen DEEPN Bildschirm einfach und kostengünstig ausgeführt werden können.

Die Y2H-Assay ist weit verbreitet für die Suche nach Protein: Protein-Interaktionen und mehrere Anpassungen und Systeme entwickelt worden. Zum größten Teil sind die gleichen Überlegungen, die helfen, Erfolg mit diesen früheren Ansätzen für DEEPN wichtig. Beispiele für wichtige Benchmarks: Ausdruck der DNA-bindende Domäne Fusionsproteine, gewährleisten einem niedrigen Hintergrund der unechten seine + Wachstum in der diploiden, enthält den Köder mit einer leeren Beute Plasmid, e...

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

Eine Hefe 2-Hybrid Bildschirm im Batch Proteininteraktionen vergleichen

Batch-Verarbeitung von Hefe-2-Hybrid-Bildschirme ermöglicht direkten Vergleich der Interaktion profile mehrere Köder Proteine mit einem hoch komplexen Satz von Fusionsproteinen Beute. Hier beschreiben wir verfeinerten Methoden, neue Reagenzien und ihre Verwendung für solche Bildschirme zu implementieren.

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 Aufrufe.  University of Iowa. Batch-Verarbeitung von Hefe-2-Hybrid-Bildschirme ermöglicht direkten Vergleich der Interaktion profile mehrere Köder Proteine mit einem hoch komplexen Satz von Fusionsproteinen Beute. Hier beschreiben wir verfeinerten Methoden, neue Reagenzien und ihre Verwendung für solche Bildschirme zu implementieren.

Serie: 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.

Ein modifiziertes Hefe-ein-Hybrid-System für Heteromere Protein-Komplex-DNA-Interaktionsstudien

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

Forschung

Biochemie

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.

Informatischen Analyse von Sequenzdaten von Batch-Hefe-2-Hybrid-Bildschirme

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

Genetik

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

Verbesserte Hefe One-Hybrid Screens, Transkriptionsfaktor Bindung an menschliche DNA-Sequenzen zu identifizieren

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

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.

Komparativen Ansatz, um die Landschaft von Wirt-Pathogen-Protein-Protein-Wechselwirkungen charakterisieren

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

Immunologie und Infektion

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-Ubiquitin Basierend Membrane Yeast Two-Hybrid (Mythos) System: Ein wirksames Instrument zur Ermittlung von Protein-Protein-Wechselwirkungen

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

Biologie

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.

Die Entwirrung der Funktion eines bakteriellen Effektor aus einem nicht-kultivierter Pflanzenschädling eine Hefe mit Zwei-Hybrid-Screen

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

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 hoher Dichte Funktionale Protein-Microarrays, um Protein-Protein Wechselwirkungen erkennen

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

Biomolekulare Interaktionsstudien mit der Hefe-Zwei-Hybrid-Technik 

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.

Autor im Rampenlicht: Verbesserte Isolierung von Interaktions-Null-Mutanten in Hefe

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.

Isolierung von Interaktions-Null-/beeinträchtigten Mutanten mit dem Hefe-Zwei-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 ...

Eine Hefe 2-Hybrid Bildschirm im Batch Proteininteraktionen vergleichen

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Ein modifiziertes Hefe-ein-Hybrid-System für Heteromere Protein-Komplex-DNA-Interaktionsstudien

Informatischen Analyse von Sequenzdaten von Batch-Hefe-2-Hybrid-Bildschirme

Verbesserte Hefe One-Hybrid Screens, Transkriptionsfaktor Bindung an menschliche DNA-Sequenzen zu identifizieren

Komparativen Ansatz, um die Landschaft von Wirt-Pathogen-Protein-Protein-Wechselwirkungen charakterisieren

Split-Ubiquitin Basierend Membrane Yeast Two-Hybrid (Mythos) System: Ein wirksames Instrument zur Ermittlung von Protein-Protein-Wechselwirkungen

Die Entwirrung der Funktion eines bakteriellen Effektor aus einem nicht-kultivierter Pflanzenschädling eine Hefe mit Zwei-Hybrid-Screen

Probing hoher Dichte Funktionale Protein-Microarrays, um Protein-Protein Wechselwirkungen erkennen

Isolierung von Interaktions-Null-/beeinträchtigten Mutanten mit dem Hefe-Zwei-Hybrid-Assay

Isolierung von Interaktions-Null-/beeinträchtigten Mutanten mit dem Hefe-Zwei-Hybrid-Assay

Isolierung von Interaktions-Null-/beeinträchtigten Mutanten mit dem Hefe-Zwei-Hybrid-Assay