4. 150 mm diameter plates should be favored as their large size
makes the 96-well plate format of the PDZome array easy to
reproduce on solid agar medium.
5. If thawing of the Y2H PDZome array on SC -Leu agar plates
has been performed with a multichannel pipette the use of the
same pipet loaded with sterile tips to scratch the clone surface
and to inoculate the 200 μL of SC -Leu liquid medium will
greatly facilitate this step.
6. Performing the mating step in 96-well plates greatly facilitates
the handling by enabling the use of multichannel pipettes.
7. Depending on the bait used for the screening, it may be useful
to optimize the temperature for both the mating and plates
growth. The optimal temperature may vary between 25
C and
30
C.
8. When using baits of several tens or hundreds amino acid long,
the occurrence of positive PDZ signals with both the WT and
truncated bait constructs may indicate the presence of an internal PDZ-binding motif within the bait.
9. 3-Amino-1,2,4-triazole (3-AT), a potent histidine analog, may
be added to the SC -Leu -Trp -His selective medium whenever
more stringent screening conditions are required in order to
reduce potential false positives. The 3-AT optimal concentration can vary substantially between yeast strains and needs to be
worked out but generally falls within the 1-25 mM range.
Acknowledgments
Through the European PDZnet consortium, the JPB and PZ
laboratories have received funding from the EU Horizon 2020
RIA under the Marie Skłodowska-Curie grant agreement
No. 675341. Work in the laboratory of PZ is currently supported
by the Fund for Scientific Research–Flanders (FWO Grants
G.0846.15 and G0C5718N), the Agence Nationale de la
Recherche (ANR-18-CE13-0017, Project SynTEV) and receives
funding from the European Union’s Horizon 2020 research and
innovation program under the Marie Sklodowska-Curie grant
agreement No 747025. JPB is a scholar of Institut Universitaire
de France.
14
Monica Castro-Cruz et al.
makes the 96-well plate format of the PDZome array easy to
reproduce on solid agar medium.
5. If thawing of the Y2H PDZome array on SC -Leu agar plates
has been performed with a multichannel pipette the use of the
same pipet loaded with sterile tips to scratch the clone surface
and to inoculate the 200 μL of SC -Leu liquid medium will
greatly facilitate this step.
6. Performing the mating step in 96-well plates greatly facilitates
the handling by enabling the use of multichannel pipettes.
7. Depending on the bait used for the screening, it may be useful
to optimize the temperature for both the mating and plates
growth. The optimal temperature may vary between 25
C and
30
C.
8. When using baits of several tens or hundreds amino acid long,
the occurrence of positive PDZ signals with both the WT and
truncated bait constructs may indicate the presence of an internal PDZ-binding motif within the bait.
9. 3-Amino-1,2,4-triazole (3-AT), a potent histidine analog, may
be added to the SC -Leu -Trp -His selective medium whenever
more stringent screening conditions are required in order to
reduce potential false positives. The 3-AT optimal concentration can vary substantially between yeast strains and needs to be
worked out but generally falls within the 1-25 mM range.
Acknowledgments
Through the European PDZnet consortium, the JPB and PZ
laboratories have received funding from the EU Horizon 2020
RIA under the Marie Skłodowska-Curie grant agreement
No. 675341. Work in the laboratory of PZ is currently supported
by the Fund for Scientific Research–Flanders (FWO Grants
G.0846.15 and G0C5718N), the Agence Nationale de la
Recherche (ANR-18-CE13-0017, Project SynTEV) and receives
funding from the European Union’s Horizon 2020 research and
innovation program under the Marie Sklodowska-Curie grant
agreement No 747025. JPB is a scholar of Institut Universitaire
de France.
14
Monica Castro-Cruz et al.