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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 2694. After boiling, chill the samples on ice and centrifuge for 30 s at 13,000g to pelletlarge cell debris. Load 10–25 µL of each sample onto a 0.1% (w/v) sodium dodecylsulfate-polyacrylamide gel.5. Prepare a Western blot and use an antibody to cI to analyze cI-fusion (bait 1)expression. Subsequently, strip the blot and probe with antibody to LexA to screenfor LexA-fusion (bait 2) expression (Note 13).3.3. Transforming a Library, and Characterizing InteractorsA partial list of available libraries compatible with the interaction trap isfound at http://www.fccc.edu/research/labs/golemis/InteractionTrapInWork.html.Currently, the majority of libraries suitable for the two-hybrid reagents describedherein are available commercially, through sources including Origene (Rockville,MD) and Invitrogen (from a noninducible promoter). If one wishes to make one’sown library, it should be cloned in a vector such as pJG4-5 or a related vector suchas pYESTRP2 (Invitrogen). The polylinker sequence at the site of cDNA insertionfor the vector pJG4-5 is shown in Fig. 3C.The protocol outlined next is designed with the goal of performing a screen,which should saturate a cDNA library derived from a genome of mammalian complexity(see also Figs. 4 and 5, for flow charts). Fewer plates will be required forscreens with libraries derived from organisms with less complex genomes, andresearchers should scale back accordingly. A protocol is provided for transformingthe library into PRT475 yeast, then using mating (24) to introduce the library againstthe bait of interest by crossing bait-containing PRT50 with library-containingPRT475 yeast. The main advantage of this approach (as opposed to directly transformingthe library into yeast containing the bait), is that if the investigator wishesto use the same library to screen multiple baits, only a single large-scale transformationis required, followed by relatively easy mating steps (see Note 14).In order to obtain a clear estimate of the frequency of cDNA-independentfalse-positives (a frequency that is important to know when deciding how manypositives to pick and characterize), it is a good idea to perform a small-scaleparallel “test mating” for new bait strains with the PRT475 yeast containingonly the library vector. This mating can be performed at the same time as thelibrary mating, and both matings can be treated identically in the next step,selecting interactors.A positive control is usually quite useful in a subsequent characterization ofpotential interactors, and can also be performed in parallel with the library transformation.Normally, these positive controls will interact with either or both of thebaits expressed in the PRT50 control strain obtained in Subheading 3.1.1., transformationb, i.e., (pEG202-Krev1 + pLacGus + pGLS22-Ras). For the experimentsdescribed herein, pJG4-5:Raf will interact with pGLS22-Ras, pJG4-5:Krit1will interact with pEG202-Krev1, and pYesTrp:RalGDS will interact with both.