FAQs Screening Service - Quick Links
- What is the price for yeast two-hybrid screening at the DKFZ?
- I don't work at DKFZ. Can I use your service?
- How long does it take?
- What do I need to prepare before using the DKFZ Y2H screening service?
- What kind of bait vectors can be used?
- Can you do the cloning for me?
- What do I get as a result of a screen?
- What if my protein cannot be screened for technical reasons?
- What is the success rate of Y2H screens?
- Customers from within DKFZ: click here
- External customers, please contact F.Schwarz.
Our priority is to support research at DKFZ. However, we are happy to screen for external customers, as long as we have free capacities, which is currently the case.
It will typically take two to three months after we receive the bait vector until we can get back to you with the analysed data.
You have to provide us with the bait, cloned in an appropriate vector. For choice of vector, see also the next paragraph.
The vector should be suitable for expression of fusion proteins with the DNA-binding domain of GAL4, and carry the TRP1 marker for selection in yeast. Typical vectors that can be used are pGBT9, pGBKT7 and pAS-2. pGBT9, or pGBT9_GW a Gateway compatible version. A small aliquot of an appropriate cloning vector can be obtained on request (contact).
Unfortunately not. Our intention is to keep the price minimal, in order to make our service affordable to academic budgets. Bait construction is time-consuming and hard to automate, resulting in relatively high costs per bait. Also, we are not better at cloning baits than the average research lab, so there is no advantage doing it within the Y2H service unit.
For these reasons, we rely on the customers to do the cloning. Also, we do not double-check bait constructs for mutations or wrong frames - so please make extra sure that the bait construct is perfect!
We send you a result sheet with the analysed data. In your report, we sum up:
- which cDNAs (gene->protein) were identified as potential interactors
- how often they were identified (the more often the better)
- in how many different cDNA libraries have they been found (the more the better)
- how many other baits did pick up the same prey
Prey proteins that have been found frequently and unspecifically are classified as "sticky", and are likely to be artefacts.
For a list of the top most promicuous human preys, see here- the strentgh (intensitiy) of the interaction signals
- the detailed data including each DNA sequence
We will highlight the interactions that we believe should be worth further examination. If you are interested, we will go through the data set together with you prey by prey!
Before screening a bait in depth, we do a test-screen in which we use conditions of varying stringency. Based on these test-screens we chose conditions which allow the isolation of hits against a clearly seperated background. However, some baits give a background that cannot be reduced even at highly stringent conditions. Other baits only yield very few hits with low intereaction signals, which disappear as soon as the stringency is slightly increased. Thus, these baits do not allow the definition of useful screening conditions.
In such cases, we recommend to cancel the screens, and will not charge the cost for the test-screens.
The success rate of two-hybrid screens is roughly as follows:
- ~ 20% of all baits cannot be screened, and the efforts are stopped after a test-screen. In this cases, no costs arise to the customer.
- 25% of baits do not give reliable data, e.g. mostly sticky preys plus a few interactors that are not found more often than once
- ~15% of baits give data that are dominated by false positives, but which also contain some specific and reproducible interactions
- Over 40% of all screens give convincing data i.e. there are few false positives and one or a group of non sticky proteins has been found repeatedly and specifically with a given bait.
Y2H screening is not a sure-fired way to scientifc success, but it is certainly worth the risk, and the effort!
