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A key part of gene functional analysis and potential drug target discovery is an understanding of how proteins interact within the cell. The two-hybrid system is a very powerful molecular genetic tool which investigates protein-protein interactions in vivo. This system can be used to study the interaction between two proteins which are expected to interact and/or and find proteins (prey) that interact with a protein you have already (bait).
Creative Biolabs provides the services on a strict fee-for-service basis. We are professional to use Gal4 and LexA based yeast two-hybrid (Y2H) systems. We are able to perform yeast two-hybrid screening against integral membrane proteins and membrane-associated/proximal proteins using a modified split-ubiquitin membrane yeast two-hybrid (MYTH) system.
The 'bait' and 'hunter' plasmids are introduced into yeast cells by transfection. In this process, the plasma membrane is disrupted to yield holes, through which the plasmids can enter. Once transfection has occurred, cells containing both plasmids are selected for by growing cells on minimal media. Only cells containing both plasmids have both genes encoding for missing nutrients, and consequently, are the only cells that will survive.
The advantages and key features of Creative Biolabs yeast two-hybrid service:
Low Rate of False Positives
We have extensive experience in handling Y2H systems marketed by Invitrogen, Clontech, Stratagene and OriGene. Also, a large number of premade Y2H cDNA libraries from these recognized companies are available and free to our customers.
High Transformation Efficiency
We use high-transformation efficient yeast strain Y190, which carries in its genome four reporter-genes, HIS 3, lac Z and MEL 1, each with distinct GAL4-responsive UASs and TATA boxes.
Experienced Technical Staff
We have more than 5 years of experience in yeast two-hybrid research work.
Step by Step Cost-Effectiveness
If the bait gene is toxic or self-active, then the customer pays only our setup fee.
Creative Biolabs optimized Y2H screening protocols. The efficiency and quality of our Y2H screening has been satisfying clients with highly reproducible and reliable results.
Detection is based on the interaction of a transcription factor (prey) with a bait DNA sequence upstream of a reporter gene. To ensure that DNA binding results in reporter-gene activation, cDNA expression libraries are used to produce hybrids between the prey and a strong trans-activating domain. Compared to biochemical techniques, the advantage of cloning transcription factors or other DNA-binding proteins via one-hybrid screenings is that the procedure does not require specific optimization of in vitro conditions.
Our combination of the one-hybrid and two-hybrid approaches increases the stringency and reliability of results obtained from two-hybrid assays alone.
The yeast three-hybrid system is important in understanding the disease process related to RNA viruses from both basic science and clinical perspective. In practice, the yeast three-hybrid system can be used to screen RNA libraries as well as cDNA libraries, though few have been reported to date since the technique is new.
BD- and AD-tagged bait and prey proteins are used. In this instance, if the query protein contains an RNA-binding activity it will co-localize the BD and AD tags and drive expression of the chosen reporter gene.
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