Protocol 3.Mating assay- large scale for hundreds of different bait or prey strains.
Materials
Freshly streaked bait and prey strains (see Protocol 1)
One set of the following 150 x 15 mm plates for each test of interactions between an activation domain-tagged protein (in a prey strain)and 96 baits (bait strains): -u-h- Glu; -w Glu; YPD; -u-h-w Glu X-Gal; -u-h-w Gal/Raf X-Gal
Replica plater and sterile velvets for 150 mm diameter plates.(A replica devise can be fashioned from a box of 200 µl pipet tips by stretching a velvet over the top of the box)
96-prong device (e.g.DanKar MC-96)with 3 mm diameter flat ended metal prongs in a 96-well configuration.Similar devices can be used in 48-well configurations for use with 100 mm plates.
0.5 to 4.0 ml sterilized tubes arranged in a 96-well configurations (e.g.cluster tubes such as Costar #4411).Ideally these tube can be capped and frozen at -80℃.
-u-h Glu liquid media,see Chapter 4
-w Glu liquid media,see Chapter 4
Sterile glycerol solution (65% (v/v)glycerol,0.1 M MgSO4,25 mM Tris-HCl pH 7.4)
Methods
1.It is most convenient to place large numbers of bait strains in a 96-well configuration (Figure 1).This can be done by inoculating 2 ml of -u-h Glu media in cluster tubes and growing to OD600 = 1.5 to 2.0.After making plates from these cultures (see step 2 below)add an equal volume of sterile glycerol solution,cap and freeze at -80℃.
2.Use the 96-prong device,sterilized in ethanol and flame,to transfer bait strains from the culture to the center of a 150 mm -u-h Glu plate.Each plate can contain 96 different bait strains.Tens of identical plates can be made from one culture.Incubate the plates at 30℃ for 48 hours or until all bait strains have grown to colonies 5 mm in diameter.These plates can be stored at 4℃ for up to 2 months and used to inoculate another liquid culture when more plates are needed.Several positions on each plate should contain control strains with baits that activate various levels of transcription (see Section 4 and Table 1).
3.Inoculate 50 ml of -w Glu liquid media with a prey strain and grow at 30℃ with shaking to OD600 = 1.5 to 3.0.Pour the culture into a sterile 150 mm plate,or into the sterile top from a box of 200 µl pipets,and use the 96-prong device,sterilized in ethanol and flame,to transfer the culture to -w Glu plates.On these plates,all 96 positions will contain the same prey strain.
4.Follow the replica plating procedure from Protocol 1 to combine the bait and prey strains to a YPD plate,and then after growth on the YPD plate at 30℃ for 24 hours,replica to X-Gal indicator,diploid selection plates (-u-h-w Glu X-Gal and -u-h-w Gal/Raf X-Gal)(see Figure 1 above).
5.Examine results after two days.
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3.Interaction mating assay with other yeast two-hybrid systems
In addition to the interaction trap,many other yeast two-hybrid systems have been developed (see Chapter 1 and Allen et al.,1995; Fields and Sternglanz,1994; Mendelsohn and Brent,1994,for reviews).All of these allow the analysis of individual protein-protein interactions,and permit interactor hunts to isolate new proteins that interact with a bait.In some instances plasmids or strains from one system can be used in another,but often the components are incompatible.Most often,the yeast selectable markers on the different components differ.In addition,systems that use Gal4 as the DNA binding domain cannot be used with yeast strains that have a wild-type GAL4 gene,and therefore,since the Gal4 protein is required to activate the GAL1 promoter,cannot be used with systems that use the GAL1 promoter to drive expression of the prey protein.Finally,use of interaction mating requires careful attention to the mating types of the strains and the selectable markers used to select the diploids.
4.Recording the results
Interaction between bait and prey results in the interaction phenotypes: growth of the .