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Yeast two hybrid assay ?


Yeast two hybrid assay: -
                                         Study interaction, detecting protein-protein interaction.

  Principle of the two-hybrid: -
A] Standard model of transcription activator: -
    - The DNA binding domain of an activator binds to an enhancer.
    - The activating domain interacted with the based complex. Recruiting it the promoter
     

B] Two hybrid assay for protein-protein interaction using gene clone technique linked the gene one protein to the part of a gene encoding a DNA binding domain, to encode 1 hybrid protein, linked the gene for another protein to the part of the gene encoding a transcription activating domain to encode a two hybrid proteins are introduce into yeast cells bearing the appropriate promoter enhancer and reporter gene (laczin) the two hybrid protein can get together and serve as an indicator activated transcription produces a bond and reporter gene product which can be detected with colorimetric assay using X-gal. (Blue colour compound)

For Example: -
 One hybrid protein contributes DNA bonding domain, and other contributes or transcription activity domain the two parts of activator are held together by the interaction between protein X and Y. if X and Y
Interact and X gal is use in the assay reporter protein gene product yeast cell will turn blue if X and Y do not interact no activator gene will occur in this case the yeast cell will remain while in the presence of X gal the gal four DNA binding domain and transcription activity possibility exist.
C] Two hybrid screen for a protein that interact with protein zinc yeast cell are transform in 2 plasmid encoding a DNA binding domain coupled to a bait protein the other is  a cell of plasmid containing many cDNA coupled to the coding region for a transcription activating domain each of this encode a fusion protein containing the activator domain fuse to and unknown DNA product each yeast cell is transform with just one of this pray encoding plasmid but several of their product are show together here for convince.
Once pray protein interact with the bait protein Z this brings together the DNA binding protein and the transcription activity domain so they can activate reporter gene.


                                                                          the yeast two-hybrid system for detecting protein-protein interaction. the target protein fused to DNA-binding domain (Bait) when this  target protein bind another specialy designed protein the cell nucleus (prey) theire interaction brings together and get transcription activate and the switches on the transcriptional reporter gene.

Reference: -



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