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268 Tikhmyanova et al.To minimize the amount of CHCl 3, use just enough to cover the colonies; try toavoid extended contact with the walls of the plate, as CHCl 3dissolves plastic.b. (Optional) Briefly rinse the plates with another approx 5 mL CHCl 3, then drainand let dry, uncovered, for another 5 min at 37°C, or for 10 min in a chemicalhood at room temperature.c. Carefully add about 10 mL of XGal- or X-Gluc agarose on the plate, makingsure that all yeast spots are completely covered. Note that it is difficult tospread less than 7 mL of agarose because the plates chill as CHCl 3evaporates.d. Put plates in 30°C incubator and keep track of color changes. Checking theplates at 20 min, 1 h, and 3 h after agarose addition is recommended. Yeastcolonies containing positive control baits (transformation c) as well as testbaits that strongly activate GusA and LacZ reporters will become dark bluecolonies in 20–60 min, whereas negative controls (transformation b)should remain as faint blue or white colonies for several hours. An optimalbait should be either comparable with the negative control or develop faintblue color.7. Next, at days 3–4 after plating, analyze the transformants for transcriptional activationof reporter genes LYS2 and LEU2, which enable growth of the transformedautotrophic yeast strain on selective media. Yeast containing both cI-and LexAfusedtest baits (from transformation a) and negative controls (from transformationb) should not grow on Gal-Raff/CM Ura − His − Leu − , Ura − His − Lys − , or Ura −His − Lys − Leu − plates (Note 8). The most important sign that baits may be suitablefor screening libraries is the absence of growth similar to negative activation control.If the tested baits grow as well as the positive control for activation (fromtransformation c) they may not be used for library screening, as they are likely toproduce high background (see Note 9).3.2.1. Detection of Bait Protein ExpressionIn general, it is recommended to evaluate the expression level and appropriatesize of the bait proteins by Western blot analysis, even if the bait is wellbehaved in the activation assays (see Note 10).1. For each bait (test and control), pick at least two primary bait/reporter transformantsfrom the Glu/CM Ura − His − master plate (see Subheading 3.2., step 5), andinoculate them into Glu/CM Ura − His − liquid medium. Grow overnight (8–12 h)on an orbital shaker at 30°C. Dilute the saturated cultures into 2 mL of the samemedium at a density of approx 0.15 OD 600, and grow at 30°C (see Note 11).2. After incubating for 4–6 h the OD 600of the cultures should reach 0.45–0.7 (measurebefore harvesting). Centrifuge 1.5 mL of each culture at 13,000g for 3–5 minin a microfuge. When each cell pellet is visible (should be approx 2–5 µL ofpacked cell volume), carefully aspirate the supernatant.3. Add 50 µL of 2X Laemmli sample buffer to each pellet, and rapidly mix by vortexingto resuspend each pellet. Boil the samples at 100°C for 5 min for immediateassay, or freeze at –70°C (in dry ice) for subsequent use (Note 12).