Custom Yeast Two-hybrid Screening Services
 

Y2H Library Screening

Creative Biolabs provides a full package of yeast two-hybrid (Y2H) library screening services for quick identification of novel binding partners for membrane or soluble proteins. Y2H screening services are also offered to map binding domains between two proteins.

We provide the services on a strict fee-for-service basis. We are professional in using Gal4 and LexA based 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. We have extensive experience in handling Y2H systems marketed by Invitrogen, Clontech, Stratagene and OriGene. Also, a large number of pre-made Y2H cDNA libraries from these recognized companies are available and free to our customers.

Our Y2H library screening service can be divided into four phases. Phase I is bait vector construction and Phase II is toxicity and self-activation test. The screening will be terminated if the bait gene is toxic to yeast or it is self-activated in yeast. Phase III is large scale library screening. Greater than 5,000,000 clones will be screened for their ability to interact with the bait and potential interactors will be identified. Importantly, we perform phenotypic validation test at Phase IV. To eliminate the possibility that the reporter phenotypes are caused by spontaneous yeast mutations or a mixture of preys during the screen, up to 20 potential interactors are isolated from yeast. The interactor plasmids are then re-transformed individually to yeast strains that express the original bait or unrelated baits to further validate the interactions. This assay is one of the most critical steps for validating specific protein-protein interactions. We also provide an optional service for interaction validation by co-immunoprecipitation in mammalian cells (phase V).

Following the screening experiments, professional Bioinformatics data analysis is available. Integrated methods are used in performing protein interaction data analysis, which including multiple sequence alignment, protein interaction database survey, global analysis of SID, pathway analysis and gene ontology analysis.

Y2H Library Construction

Creative Biolabs also offers quality yeast two-hybrid library construction services. cDNA library or genomic DNA library (for bacteria) construction is an integrated part of our all-in-one yeast two hybrid platform. If there is a regular cDNA library [particular normalized one] or genomic DNA library available, we can take the library, extract the DNA inserts and then subclone them into a Y2H vector. Alternatively, we can start from total RNA or mRNA for cDNA library construction. We reverse-transcribe the mRNAs into cDNAs and clone them into a proper vector. The primary library will have at least 3 x 10⁶ primary variant with average insert length 0.5-3kb, which should be far more enough to represent every gene for most organisms.

Y3H Library Screening

Creative Biolabs uses Clontech pBridge Three-Hybrid Vector in combination with its Matchmaker Yeast Two-Hybrid System to offer yeast three-hybrid [Y3H] library screening services.

Y1H Library Screening

Using Clontech’s Matchmaker Gold Yeast One-Hybrid (Y1H) Library Screening System, we also provide quality services for identifying and characterizing novel protein-DNA interactions. The system employs Aureobasidin A (AbA; 1) selection, which provides the most stringent yeast one-hybrid screening strategy available.