4. Incubate the conjugating cells for 5–6 h at 30
C.
5. Spin down the cells by a brief (2–3 min) centrifugation at
medium speed in a microcentrifuge.
6. Wash the cells once with 1 mL of sterile ddH 2 O before resuspending the cell pellet in 100 μL of sterile ddH 2 O.
7. Spot 5 and 25 μL of the cell suspension on both SC -Leu -Trp
and SC -Leu -Trp -His solid media.
8. Test for growth after a 2–3 day of incubation at 30
C.
9. Growth on SC -Leu -Trp medium is a readout for the mating
efficiency while, as already discussed in step 1, the ability to
grow on SC -Leu -Trp -His medium directly reflects the bait
tendency to self-activate GAL1-HIS3 transcription.
3.6 Yeast
Two-Hybrid Screening
of PDZome Array
Each clone of the human PDZome library used in this chapter has
been subcloned in pACT2-GW before being transformed in the
Y187 yeast strain and expanded in a three 96-well plate format
(Fig. 2). A frozen stock of this Y2H PDZome resource has been
made, and here we describe the successive steps when implementing a new screen of the resource (see Note 3).
1. Thaw the frozen Y2H PDZome array by placing the three 96w
stock plates on ice for 30 min.
2. Duplicate the Y2H PDZome library with a multichannel
pipette by spotting 5 μL of each clone on SC -Leu agar medium
(see Note 4).
3. Incubate the plates at 30
C until all spots exhibit robust
growth. This step generally takes 2–3 days but may take a few
more days. You may now proceed with the screening as illustrated in Fig. 3 and as detailed below.
4. Day1, distribute 200 μL of SC -Leu medium per well in three
96-deep-well plates and duplicate the Y2H PDZome by inoculating each well with a Y187 PDZ clone from the agar plates
obtained in step 3 (see Note 5).
5. Seal the plates with an adhesive aluminum foil and let the cells
grow for 2 days at 30
C under vigorous agitation.
6. Day2, inoculate 8 mL of SC-Trp liquid medium with baitbearing AH109 strains (WT and PBM mutant) and incubate
overnight at 30
C under agitation.
7. Day3, dispense 150 μL of YPD complete medium per well in
three 96 well plates (see Note 6).
8. Distribute in each well 20 μL of the bait-containing AH109 cell
culture obtained in step 6.
Y2H Screening of PDZ Interactions
11
C.
5. Spin down the cells by a brief (2–3 min) centrifugation at
medium speed in a microcentrifuge.
6. Wash the cells once with 1 mL of sterile ddH 2 O before resuspending the cell pellet in 100 μL of sterile ddH 2 O.
7. Spot 5 and 25 μL of the cell suspension on both SC -Leu -Trp
and SC -Leu -Trp -His solid media.
8. Test for growth after a 2–3 day of incubation at 30
C.
9. Growth on SC -Leu -Trp medium is a readout for the mating
efficiency while, as already discussed in step 1, the ability to
grow on SC -Leu -Trp -His medium directly reflects the bait
tendency to self-activate GAL1-HIS3 transcription.
3.6 Yeast
Two-Hybrid Screening
of PDZome Array
Each clone of the human PDZome library used in this chapter has
been subcloned in pACT2-GW before being transformed in the
Y187 yeast strain and expanded in a three 96-well plate format
(Fig. 2). A frozen stock of this Y2H PDZome resource has been
made, and here we describe the successive steps when implementing a new screen of the resource (see Note 3).
1. Thaw the frozen Y2H PDZome array by placing the three 96w
stock plates on ice for 30 min.
2. Duplicate the Y2H PDZome library with a multichannel
pipette by spotting 5 μL of each clone on SC -Leu agar medium
(see Note 4).
3. Incubate the plates at 30
C until all spots exhibit robust
growth. This step generally takes 2–3 days but may take a few
more days. You may now proceed with the screening as illustrated in Fig. 3 and as detailed below.
4. Day1, distribute 200 μL of SC -Leu medium per well in three
96-deep-well plates and duplicate the Y2H PDZome by inoculating each well with a Y187 PDZ clone from the agar plates
obtained in step 3 (see Note 5).
5. Seal the plates with an adhesive aluminum foil and let the cells
grow for 2 days at 30
C under vigorous agitation.
6. Day2, inoculate 8 mL of SC-Trp liquid medium with baitbearing AH109 strains (WT and PBM mutant) and incubate
overnight at 30
C under agitation.
7. Day3, dispense 150 μL of YPD complete medium per well in
three 96 well plates (see Note 6).
8. Distribute in each well 20 μL of the bait-containing AH109 cell
culture obtained in step 6.
Y2H Screening of PDZ Interactions
11