Principal teacher: Ilya Serebriiskii

Two types of classes are available: 

a basic yeast two-hybrid class and an advanced class.

Please note, each student on the class will have access to  all reagents 

needed to start working with the Yeast Two-Hybrid and/or Bacterial Two-Hybrid Systems!

See "extras" below. No extra charge, but MTA may be required.


 Class I
click for typical schedule
 Class II
schedule under development
Lectures

  • Introduction to the yeast two-hybrid system 

  • Yeast as a Genetic Tool 

  • Two-hybrid system components 

  • The interaction-trap system as an example 

  • Bait Design, Cloning & Assessment. Troubleshooting 

  • Library Screening 

  • Analysis of candidates and second confirmation of positives

    Interaction mating, beta-gal assays and related techniques

  • False Positives. YTH on the Internet

  • Variations & New Developments
    (one-hybrid, three-hybrid etc)  

    Dual Bait (introduction) and YTH review

    How to choose your working system

  • Non-transcription based two-hybrid systems: Split-ubiquitin & Ras Recruitment Systems

  • Bacterial two-hybrid systems

  • Mammalian two-hybrid systems

  • In-depth comparison of advanced YTH systems

  • Specialized yeast two-hybrid systems (one, 1.5, tri-hybrid, RNA etc.)

  • Reverse yeast two-hybrid systems

  • Combined yeast-bacterial system based on Dual Bait

  • Advanced study of False positives

  • Rational Bait Design

  • Bioinformatics: how to extend your clone in silico

  • Bioinformatics: virtual two-hybrid screening and visualization of protein-protein interactions (Cytoscape)
Laboratory

  • Mating assay

  • Yeast transformation

  • Bait Assessment

  • Replica technique

  •  ß--gal assay techniques: on filters, on plates and by CHCl3 overlay

  • ß--gal assay in liquid

  • Yeast DNA prep and PCR


 

  • Advanced techniques in mating: velveteen vs. replicator

  • LexA-based vs Gal4-based systems: side by side comparison.

  • Reverse yeast two-hybrid system: counterselection assay

  • Detections of False Positives

  • High-throughput ß-gal assay in liquid using 96-well plates

  • Split ubiquitin two-hybrid system 

  • Bacterial two-hybrid systems: - growth and colorimietric assays

  • Mammalian two-hybrid system

  • Dual Bait: simultaneous two-color assay
Extras

  • A complete set of second-generation reagents to start two-hybrid screening (compare content  to Origene’s set DKT100 at $1800.00)

  • An aliquote of the antibody to test bait expression by Western blot (compare to Invitrogen’s R990-25 at $170.00)*

  • A complete protocol for performing two-hybrid screening (compare to Humana Press eBook chapter at $30.00)








* upon cloning of your gene of interest into bait plasmid

  • A complete set of reagents to start Dual Bait yeast two-hybrid screening (compare to Invitrogen’s Hybrid Hunter at $640.00

  • Two aliquotes of the antibodies to test baits expression by Western blot (compare to Invitrogen’s at $170.00 each)*

  • A complete protocol for performing Dual Bait two-hybrid screening (compare to Humana Press eBook chapter at $30.00)

  • A set of vectors to start bacterial two-hybrid system (compare to BacterioMatch Vector Kit from Stratagene at $587.00)

  • A chance to earn (the best student will get it!)
     Prot-prot interaction manual Protein-Protein Interactions: A Molecular Cloning Manual
    Edited By Erica A. Golemis,
     Fox Chase Cancer Center



  • © 2002 • 682 pp., illus., appendices, index
    Hardcover • $205 • ISBN 0-87969-604-4

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