- You need new insights into the function of your protein?
- You are out to compare interaction patterns of mutated and wild type proteins?
- You are interested in finding weak or transient interactions?
We offer a well established high-throughput yeast two-hybrid (Y2H) screening service providing screening libraries of various tissues and two human OFR libraries (N- and C-terminal constructs with >10,000 ORFs each).
In over 2,500 screens with >700 different bait proteins/protein-fragments accomplished thus far we have obtained broad experience in practice as well as in the interpretation of the results.
Yeast Two-Hybrid (Y2H) screening is a well-established method to detect protein protein interactions (see here for a powerpoint explanation of the basic method).
The DNA binding domain (DBD) of the transcription factor is expressed as a hybrid protein fused to the protein of interest (marked "B" as 'bait', see top left in the picture), and the activation domain (AD) is fused to another protein ("P" as 'prey', see top right in the picture).
If an interaction between bait and prey takes place the activation domain will come into the proper position to activate a reporter gene. In an Y2H screen a bait protein is tested against a complex collection of proteins or protein fragments originating from cDNA or ORF libraries (fusions with the AD). Interacting proteins (preys) are identified by sequencing the corresponding plasmid DNA. We offer more than 30 different cDNA screening libraries from several species and tissues and two human OFR libraries (N- and C-terminal constructs with >10,000 ORFs each).
For frequently asked questions, please see our FAQ-list.
How to start
The best way is to send us Gene Symbol, Protein Name or Accession of the proteins planned to screen. We will try to give an estimate for each protein if it could be a suitable candidate for Y2H-screening. We will query our screening database and try to get hints about appropriate libraries, protein fragments and possible tendency for promiscuity. Of course, we will treat your data confidentially. Please use the contact form right hand.
What we need from you
We need your bait DNA cloned into a plasmid suitable for our system, such as pGBT9 or pGBKT7 or a Gateway-compatible variant. There are ntermial and also cterminal versions available. For detail information - please request. We require 10 µg DNA of your bait construct at a concentration > 100ng/µl.
Gateway Shuttling Service
On request we can offer to perform standard Gateway Shuttling of insert DNA into appropriate Destination vectors at a moderate price. You supply a sequence verified Entry clone or Entry vector plasmid (mini prep) and we will shuttle the insert in an appropriate bait Destination vector. We additionally have access to a huge mammalian (mainly human) full-ORF clone collection. We can check if appropriate clones harbouring your gene of interest are available. We can use our full ORF-clones for bait cloning, but please note: we are not allowed to send the original Entry clones to external customers.
Please use the contact form (right column on top)
What we do
The yeast two-hybrid screening service offers protein interaction screens with an automated high-throughput Y2H screening procedure. We
- Optimise screening conditions for your bait
- Screen libraries of your choice
- Retrieve the library inserts of positive clones by PCR
- Sequence the inserts from the 5' end
- Analyse the sequence by blast analysis
- Compare the results to previous screens
- Prepare a report for you
What we return
You will receive a report from us highlighting interacting proteins that qualify as high-confidence interactors based on comparisons with previous screens. You can also get the PCR products 'harbouring' the library inserts on request. We generally do not keep the positive yeast cells.
For more details regarding the two-hybrid service, cloning vectors, experimental planning support, or prices for external customers, please contact Sara Burmester. Information on pricing for internal customers can be found at the GPCF pricelist.