Medicilon’s Yeast Two Hybrid Services

 The yeast “two hybrid” or “interaction trap” assay, is a technique used to study protein-protein interactions, which are critical in virtually all cellular processes. The study of protein-protein interactions can be divided into three major parts:

• Identification of binding proteins
• Characterization of known interactions
• Potential to manipulate such interactions

This technology is based on the fact that the activating domain (AD) and binding domain (BD) of a transcription factor can be separated and still function when they get into close proximity. Y2H screening is therefore designed to use a BD fused bait protein to screen against a library of AD fused prey proteins. The bait is usually a known protein that the researcher tries to identify new interacting partners with. The prey is usually a library of proteins to be screened from. When the bait protein binds to the prey protein, transcription of the specially designed reporter genes occurs and allows selection of this positive clone. Medicilon use modified yeast strains which incorporates several reporter genes to reduce false positives. We also use different GAL4 binding elements to increase sensitivity of the assay.

Yeast Two-Hybrid Procedure

1. Generate a GAL4 DNA-BD fusion by cloning the gene of interest in frame with the GAL4 DNA binding domain of pGBKT7.
2. Transform AH109 with bait plasmid, detect bait expression and test for autoactivation and cell toxicity.
3. Mate the pretransformed library strain with the bait strain.
4. Restreak colonies that can grow on TDO plates to QDO plates.
5. Restreak positive clones to single colonies on QDO/+X-a-Gal plates.
6. Rescue plasmid DNA from positive yeast clones.
7. Identify and isolate AD/library plasmids from E. coli transformants.
8. Cotransform DNA-BD/bait and AD/library plasmids into AH109.
9. Repeat Step 5.

10. Select true positive clones and sequence cDNA inserts.

Our Advantages:

• Highly reduced rate of false positives
• Short turn-around time
• Best price in the market
• Highly customized service

Medicilon offers a wide range of ready-to-use prey protein cDNA libraries. We can also custom develop homogenized cDNA libraries according to your specific requirements. Screening and validation process can be done in as short as two months! We also provide co-immunoprecipitation service to validate your result from the Y2H screening.

Tips for Two-Hybrid Screening
Minimizing False Positives:
Run replicates of the experiment to reduce the likelihood of indiscriminate reporter gene activation. Including a prey-only control also provides a baseline level of reporter activation.

Vary the expression levels of bait and prey proteins – overexpression can lead to false interactions. Lowered expression levels will increase the stringency of your binding.

And most importantly: Independently validate identified binding partners with other techniques!

 

Minimizing False Negatives:

Interactions leading to detectable reporter signals depend on a number of factors, including protein expression and correct folding, post-translational modification, protein degradation, access to the nucleus in eukaryotic screens and fusion configuration. The possibility for problems with one or more of these parameters can lead to a high number of false negatives in Y2H screens.

Testing your reporter system with a pair of proteins that are known to bind serves as a good positive control to ensure that your setup works. The last thing you want is to run your lovely library screen only to find the system itself is not working because your reporter protein doesn’t function in yeast!

Many false negatives stem from or are exacerbated by expression of target proteins in heterologous systems .

While this may be difficult to resolve for a wide range of prey proteins, there are now a number of two-hybrid systems available in model organisms, including bacteria, alternative fungi and mammalian cells.

If the problem is that your proteins are not post-translationally modified, co-expression of the enzyme responsible for the modification in the assay host strain can help.

In order to reduce the chance that the configuration of your fusion proteins physically blocks the binding sites for the protein partner or the UAS/reporter gene, the same bait and prey libraries can be screened using both N- and C-terminal fusions of these proteins. This way both ‘ends’ of your protein are screened for binding.

Using a variety of expression levels and systems can alleviate false negatives that arise from low or faulty expression of your target proteins. In fact, a good overall strategy to reduce false negatives and produce a more vigourous screen is to use a number of different bait and prey vectors. This has been shown to be as effective as using five independent protein interaction detection methods.

In order to bind DNA and activate a reporter gene in eukaryotic cells, fusion proteins must gain access to the nucleus. To circumvent this requirement for transmembrane proteins, a split ubiquitin system has been devised.

Contact Us

Email : marketing@medicilon.com

Tel : +86 021 58591500

Tips : Above is part of yeast two hybrid analysis, yeast 2 hybrid service and yeast two hybrid screen. You can also CONTACT US with any question or enquiry you may have. We will be happy to discuss your needs in detail and design an appropriate plan of action.