Because our lab is particularly interested in the functions of NF-Y proteins and we have developed Arabidopsis Y2H interaction datasets for several of these proteins, we decided to use their Brachypodium orthologs for initial proof-of-concept screening.
Therefore, using NF-YC as the bait protein fulfilled our criteria for good proof-of-concept screening material.
In keeping with a small molecule pharmacological approach, we describe here a novel screening strategy optimized for the detection of caspase-8 pathway-specific TRAIL sensitizers, and demonstrate its usefulness by a proof-of-concept screen.
The best protein choice for initial proof-of-concept screens is one with well-described interacting partners.
Proof-of-concept screens using the two Y2H libraries demonstrate that expected interactors can be readily isolated against a known bait.
Initial proof-of-concept screens demonstrated that a protein with known interaction partners could readily re-isolate those partners, as well as novel interactors.
Because Bradi3g05270 did not display any obvious autoactivation, we chose this protein to perform the actual proof-of-concept screens against the Brachypodium libraries.
We demonstrated the utility of this sample introduction method combined with MCE for HTS by performing a proof-of-concept screen with PKA and a set 140 small molecules.
Because our lab is interested in the functions of NUCLEAR FACTOR Y (NF-Y) transcription factors [ 27] and interactors are already described for a subset of these proteins [ 28- 30], we isolated a Brachypodium NF-Y ortholog and used this as "bait" to perform initial proof-of-concept screens with both Y2H libraries.
Acceptable Z' factors were achieved (>0.5) in 384 well format, and proof of concept screens of a small library enriched in bioactive compounds demonstrated acceptable hit rates.
As proof-of-concept, we screened the M2 population for induced mutations in two genes related to fatty acid biosynthesis, FatA an acyl-ACP thioesterase and SAD the stearoyl-ACP desaturase and identified a total of 26 mutations.
