Using the Brent 2-Hybrid System I have so far successfully cloned
by bait into the DNAbd plasmid, transfected EGY48 w/ pSH18-34, checked
for self reactivity, done a western to detect presence of the fusion
protein, proceeded to transfect lib.plasmid containing a Hela cell lib,
made glycerol stocks and proceeded to screen for protein interaction by
plating onto Gal/Raf -His, -Ura, -Trp, -Leu plates. For curiosity I also
plated the same cells onto Gal/Raf -H,-U,-T and Glu -H,-U,-T. The yeast
colonies that grew the next day were reddish in color, where as the yeast
on the Glu -H, -U,-T were white and present in high density as expected.
Most interesting was the Gal/Raf -H,-U,-T plate contained a lawn of white
colonies as well as a smaller number of large reddish colonies. In all
the plates the number of reddish colonies was the same so I assumed that
these were in fact yeast that were growing on -Leu plates because of some
relevant protein interaction. Subsequently I have tested for Gal
dependence and no color change was observed. What is going on? Can
anyone help to explain these results? I would be very grateful.
Kerry
