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The principle of Y2H
I N the few years since its introduction, the two-hybrid systemhas proven invaluable for identifjmg interactions between proteins. It has been used to establish physical interactions between genetically identified partners , identify contacts among the subunits of multiprotein complexes ,and to map the specific domains within proteins responsible for an interaction . Most importantly, the two-hybrid system has allowetdh e identification of new interacting proteins by screening expression libraries .The two-hybrid system utilizetsw o plasmid-borne gene fusions that are cotransformed into a host yeast strain containing inducible reportegr enes. The protein of interest, or “bait”, is encoded as a gene fusion to a DNA binding domain from either the GAL4 or L e d protein. A second protein, or a library of proteins, is encoded as a gene fusion to a transcription activation domain. Interaction between the two proteins results in the localization of the transcription activation domain to the DNA of the host strain, activating transcription of the adjacent reporter genes and generating a phenotypic signal.
What Can I Do With My Very Own Yeast Two-Hybrid??
Firstly, the yeast two-hybrid assay can identify novel protein-protein interactions. By using a number of different proteins as potential binding partners, it is possible to detect interactions that were previously uncharacterized3. Secondly, the yeast two-hybrid assay can be used to characterize interactions already known to occur. Characterization could include determining which protein domains are responsible for the interaction, by using truncated proteins, or under what conditions interactions take place, by altering the intracellular environment.
The last and most recent application of the yeast two-hybrid involves manipulating protein-protein interactions in an attempt to understand its biological relevance. For example, many disorders arise due to mutations causing the protein to be non-functional, or have altered function. Such is the case of some cancers; a mutation in a pro-growth pathway does not allow for the binding of negative regulatory proteins, resulting in the pro-growth pathway never turning off. The yeast two-hybrid is one means of determining how mutation affects a proteins interaction with other proteins. When a mutation is identified that affects binding, the significance of this mutation can be studied further by creating an organism that has this mutation and characterizing its phenotype.
The yeast two-hybrid assay is an eloquent means of investigating protein-protein interactions. A fairly new addition to the family of biological studies, these interactions have become increasingly important to our understanding of biological systems in the past few years. While isolation of a protein in an attempt to understand its function still has its place in biological research, we now understand that biological reactions do not occur in isolation. A protein is constantly interacting with other proteins in what we now know to be a delicate balance — to ignore this wealth of information would be to deny ourselves the opportunity to fully appreciate the stuff that life is made of.
1 Yeast 2 hybrid cDNA library construction
Construct special gene library library for customer. The tissue,cell or cDNA for library construction should be supplied by customer.
Time required: 2-3 months
2 To screen a cDNA library by yeast 2 hybrid
To screen the designated ed cDNA library for customers by yeast two hybrid, so as to find a gene interacted with the bait gene. Customers provide the bait gene and the cDNA library.
Time required:three months.
After the screening is completed, we will provide specific reports and positive gene clone to customers.
3 To detect the interaction between 2 genes by Y2H
To detect interactions between two genes or more than one pairs of genes by yeast two hybrid system for customers. Customers provide the gene clone.
Period: one months.
Once the experiment is completed, we will provide specific reports and yeast strain cotransformationed with interacted pairs of genes to customers.
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