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306 Serebriiskii et al.1. From the NM_AC master plate, inoculate at least two colonies per test bait into 2 mLof NM_AC liquid medium (if using an LB_AC master plate, adjust to LB_AC). Inparallel, set up cultures of pBaitC-Ras and/or pBaitC-zip transformants as positivecontrols for protein expression (see Fig. 5, lanes 1 and 5). Grow overnight cultures onan orbital shaker at 37°C. For optimal protein expression, dilute the saturated cultures1/100 into fresh tubes containing 2 mL of the same medium with or without IPTG toobtain exponentially growing cultures (see Note 14).2. After the OD 600nm of the cultures reaches 0.35–0.5 (after about 4–6 h), harvestcells from 1.5 mL of each culture by centrifuging at 13,000g for 3–5 min in amicro-centrifuge. Carefully remove supernatant from the cell pellet.3. Add 50 µL of 2X Laemmli sample buffer to each tube, and rapidly vortex to resuspendeach pellet. Heat the samples at 100°C for 5 min for immediate assay, orfreeze (using dry ice for flash freezing) and store at –70°C for subsequent use:frozen samples should then be heated at 100°C before proceeding to step 4.4. After heating, chill the samples on ice, then centrifuge for 30 s at 13,000g to pelletlarge cellular debris. Dilute 1:100 in 1X Laemmli sample buffer, and load 10–25 µLof each sample onto a 10% (w/v) sodium dodecyl sulfate polyacrylamide gelelectrophoresis gel.5. Prepare for Western blot analysis using standard transfer approaches (17), andprobe membrane using an antibody to cI. This allows comparison of expressionlevels of the bait protein under test with control cI-fused proteins (see Fig. 5 for atypical example of an immunoblot detecting cI-bait expression).3.1.4. Troubleshooting Baits With Undesirable Characteristics1. If a bait is expressed at inappropriately low/high levels (more than 10-fold divergentfrom the controls), one may wish to consider adjusting the concentration ofIPTG used in the test plates.2. If a bait autoactivates the reporter to any significant degree, it is probably worthwhileto subdivide the bait into overlapping domains, creating new baits that mayhave reduced autoactivation potential. In doing so, try to divide the bait accordingto any available information about protein structure, in order to avoid disruptingdiscrete domains.3.2. Introducing the Library Into the Selection Strainand Selecting InteractorsMethods for introducing prey libraries into the selection strain cells differdepending on the library. In the most straightforward approach, the preyplasmidlibrary is electroporated directly into high-transformation efficiencycells along with the pBait plasmid. Alternatively, a library constructedusing pLibB can be converted into a library of infectious transducingphage, and subsequently introduced into selection strain cells containingonly the bait (prepared in Subheading 3.1.1., see Note 11) by simpleinfection with the phage.