Hope for the best...

because the Interaction Trap yielded on average four times more successes than the failures. But you have to know when prepare to the worst!

Major traps for a hunter

In our opinion, the two biggest dangers preventing success of the two-hybrid hunts are transactivation by the bait protein itself and failure to express the bait properly. For these obvious reasons a few researchers abandoned hunts without even starting a screen. Among about analyzed 115 library screenings, 16 were reported to be total failures (i.e. either no positives, either only false positives). These cases can be tentatively explained as follows:

• 4 colleagues experienced difficulties with protein expression (i.e. no protein was detected on Wester blots);
• 3 failures could be due to weak transactivation by the bait protein;
• in 2 cases, a number of primary transformants too low to represent genome complexity is suspected;
• in one case the transformants were plated directly on selective medium without prior plating on UHW medium;
• finally, 2 screening were performed with all possible precations, and no obvious reason can be evoked to explain the failure in these cases. However, it can be either the absence of the alleged interactor in the library, or even more profound biological reasons.
• 4 colleagues were sufficienly disappointed that they did not answer the questions which could give us clue on the possible reasons of these particular failures

Your chances of success

Analysing the bulk of the reported screenings, and assuming that our database is representative, we can conclude that:

• if your protein is properly expressed and is not activating either of the reporters, odds are 6 to 1 that you will pull out something which makes biological sense, if you screen an adequate number of clones.
  Note: for human, genome complexity will be represented by approx. 1-2 million primary transformants; for Drosophila, by ???.
• if your protein is weakly transactivating, your chances drop, but not really dramatically. However, if your protein is strongly activating leu reporter, we would recommend refraining from plating directly on X-Gal plates. Three colleagues attempted, and none succeeded.
• if you cannot detect your bait protein in yeast, your chanses drop substantially. Consider the situation once again before venturing on a screen. As in case with activating baits, you can spend a lot of time for nothing.

Condi- tions	No activation, protein expressed	Weak activation or leu activation	Problems with the protein expression
red: + success blue: - failure green: ? ongoing
Total:	57	18	10

The degree of your success can vary!

The complexity of many genomes, and the complexity of the web of protein interactions is still beyond of the abilities of any human-made systems. So, you will find not necessarily what you want to find; rather, it is better not to to be ruled by preconceptions and to be aware of the limitation of the system. This can be illustrated by following data:

of 64 successful hunts, the partners were known for 30 baits before screenings.

However, only 11 of these baits found previously known partners, whereas 19 baits only identified novel, albeit biologically relevant partners.

This analysis emphasizes that the two-hybrid systems are the tools to uncover unanticipated interactions!