X

Download The Yeast Two Hybrid System The Jena Protein Protein PowerPoint Presentation


Login   OR  Register
X

Share page



  Preview

               
Home / Health & Wellness / Health & Wellness Presentations / The Yeast Two Hybrid System The Jena Protein Protein PowerPoint Presentation

The Yeast Two Hybrid System The Jena Protein Protein PowerPoint Presentation

worldwideweb By : worldwideweb

On : Jan 08, 2015

In : Health & Wellness

Embed :
663
views

0
downloads
Login / Signup - with account for


  • → Make favorite
  • → Flag as inappropriate
  • → Download Presentation
  • → Share Presentation
  • Slide 1 - The Yeast Two-Hybrid System Anne C. Luebke
  • Slide 2 - What is the yeast two-hybrid system used for? Identifies novel protein-protein interactions Can identify protein cascades Identifies mutations that affect protein-protein binding Can identify interfering proteins in known interactions (Reverse Two-Hybrid System)
  • Slide 3 - How does it work? Uses yeast as a model for eukaryotic protein interactions A library is screened or a protein is characterized using a bait construct Interactions are identified by the transcription of reporter genes Positives are selected using differential media
  • Slide 4 - The Model DNA-Binding Domain Bait Protein Prey Protein Transcription Activating Region Reporter Gene DNA-Binding Site
  • Slide 5 - Steps to Screen a Library Create the Bait Plasmid Construct from the gene of interest and the DNA binding domain of Gal4 or LexA or other suitable domain Transform with the bait construct a yeast strain lacking the promoter for the reporter genes and select for transformed yeast Transform the yeast again with the library plasmids Select for interaction
  • Slide 6 - Sequence analysis Isolate plasmid from yeast and transform E. coli Purify plasmid from E. coli and sequence Blast sequence against database for known proteins or construct a possible protein sequence from the DNA sequence and compare to other proteins
  • Slide 7 - Reporter Genes LacZ reporter - Blue/White Screening HIS3 reporter - Screen on His+ media (usually need to add 3AT to increase selectivity) LEU2 reporter - Screen on Leu+ media ADE2 reporter - Screen on Ade+ media URA3 reporter - Screen on Ura+ media (can do negative selection by adding FOA)
  • Slide 8 - Plasmid Constructs Plasmids are constructed with the Gal4 DNA binding domain (or other suitable domain) in front of a Multiple Cloning Site (MCS) The plasmid contains genes that can be used for selection such as Amp, Leu2, Ura3, or Trp1
  • Slide 9 - Sample Plasmid From Golemis Lab Homepage
  • Slide 10 - False Positives False positives are the largest problem with the yeast two-hybrid system Can be caused by: Non-specific binding of the prey Ability to induce transcription without interaction with the bait (Majority of false positives)
  • Slide 11 - Elimination of False Positives Sequence Analysis Plasmid Loss Assays Retransformation of both strain with bait plasmid and strain without bait plasmid Test for interaction with an unrelated protein as bait Two (or more) step selections
  • Slide 12 - Advantages Immediate availability of the cloned gene of the interacting protein Only a single plasmid construction is required Interactions are detected in vivo Weak, transient interactions can be detected Can accumulate a weak signal over time
  • Slide 13 - Examples of Uses of the Yeast Two-Hybrid System Identification of caspase substrates Interaction of Calmodulin and L-Isoaspartyl Methyltransferase Genetic characterization of mutations in E2F1 Peptide hormone-receptor interactions Pha-4 interactions in C. elegans
  • Slide 14 - References Bartel, Paul, C. Chien, R. Sternglanz, S. Fields. “Elimination of False Positives that Arise in Using the Two-Hybrid System.” Biotechniques (1993) Vol. 14, no. 6, p. 920-924. Chien, Cheng-ting, P. Bartel, R. Sternglanz, S. Fields. “The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest.” Proc. Natl. Acad. Sci. USA (1991) Vol. 88, p. 9578-9582. Fields, Stanley, O. Song. "A novel genetic system to detect protein-protein interactions." Nature (1989) Vol. 340, p.245-246. James, Philip, J. Halladay, E. Craig. "Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast." Genetics (1996) Vol. 144, p. 1425-1436. Kamada, S, H. Kusano, H. Fujita, M. Ohtsu, R. Koya, N. Kuzumaki, Y. Tsujimoto. "A cloning method for caspase substrates that uses the yeast two-hybrid system: Cloning of the antiapoptotic gene gelsolin." Proc. Natl. Acad. Sci. USA (1998) Vol 95, p. 8532-8537. O'Connor, Mirriam, C. O'Connor. "Complex Interactions of the Protein L-Isoaspartyl Methyltransferase and Calmodulin Revealed with the Yeast Two-hybrid System." The Journal of Biological Chemistry (1998) Vol. 273, p. 12909-12913. Staudinger, Jeff, J. Zhou, R. Burgess, S. Elledge, E. Olson. "PICK1: A Perinuclear Binding Protein and Substrate for Protein Kinase C Isolated by the Yeast Two-Hybrid System." The Journal of Cell Biology (1995) Vol. 128, p. 263-271.
  • Slide 15 - References continued Vidal, Marc, P. Braun, E. Chen, J. Boeke, E. Harlow. "Genetic Characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system." Proc. Natl. Acad. Sci. USA (1996) Vol. 93, p. 10321-10326. White, Michael. "The yeast two-hybrid system: Forward and reverse." Proc. Natl. Acad. Sci. USA (1996) Vol 93, p. 10001-10003. Zhu, Jianwei, C. Kahn. "Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system." Proc. Natl. Acad. Sci. USA (1997) Vol. 94, p. 13063-13068. Lab of Erica Golemis http://www.fccc.edu/research/labs/golemis/EG_homepage.html Special thanks to Dr. Susan Mango and the University of Utah
ECG Heart Beat Free PowerPoint Template

ECG Heart Beat

Views : 1646

Back to School Free PowerPoint Template

Back to School

Views : 506

Yoga Meditation Free PowerPoint Template

Yoga Meditation

Views : 754

Morning Running Free PowerPoint Template

Morning Running

Views : 771

AI Free PowerPoint Template

AI

Views : 660

Wedding Cake Free PowerPoint Template

Wedding Cake

Views : 805

Dentist Free PowerPoint Template

Dentist

Views : 376

Butterfly Free PowerPoint Template

Butterfly

Views : 642

Stop Aids Free PowerPoint Template

Stop Aids

Views : 527

Drone Free PowerPoint Template

Drone

Views : 4100

Global Warming Pollution Free PowerPoint Template

Global Warming Pollution

Views : 873

Autumn Free PowerPoint Template

Autumn

Views : 563

Global Warming Free PowerPoint Template

Global Warming

Views : 550

Agriculture Free PowerPoint Template

Agriculture

Views : 904

Running Track Free PowerPoint Template

Running Track

Views : 426

Burger Free PowerPoint Template

Burger

Views : 558

Photography Free PowerPoint Template

Photography

Views : 448

Heart Free PowerPoint Template

Heart

Views : 484

Vegetables Free PowerPoint Template

Vegetables

Views : 506

Gerbera Free PowerPoint Template

Gerbera

Views : 336

Description : PowerPoint presentation on The Yeast Two Hybrid System The Jena Protein Protein, download now ppt of The Yeast Two Hybrid System The Jena Protein Protein

Tags : The Yeast Two Hybrid System The Jena Protein Protein

Shortcode : Get Shareable link