2. Did you screen with a library or examine interactions between defined sets of proteins ?
3. What kind of protein did you use?
transcription factor, kinase, G-protein,
other? please specify!
If you are willing, what is the actual name of the protein?
4. How large was the bait used (not including the fusion partner)? AA or kD
Was this a full-length protein or a truncation?
5. Where is the protein normally known to localize within the cell?
nucleus, ER, Goldgi, cytoplasm, membrane,
6. Did the bait have any residual ability to activate transcription of either the LEU2 or the LacZ reporters prior to library screen?
If Yes, how (qualitatively) did it compare to pSH17-4 control? did not compare, much weaker, about the same, stronger.
7. What species was the bait derived from?
humans, mouse, rat, C.elegance, S.cerevisiae, Drosophila,
8. What combination of yeast strain and LacZ reporter plasmid was used for screening?
9. Did you try to confirm that your fusion protein was made and the correct size by Western blot prior to commencing screening?
No, did not try Yes, and it looked good Yes, but protein was degraded/funky
10. What library was used for screening?
11. Approximately how many primary yeast transformants were obtained for screening?
x106
How many cells (or colony forming units), approximately, were then plated to selective medium? x106
12. Following plating on selective medium, how many colonies came up as prospective positives? On what days (range) after plating did they arise? How many of these positives did you proceed to analyse?
13. Did initially selected positives strongly activate both LEU2 and LacZ reporters, was there bias of one over the other? (see table below)
Did you obtain what you believe is potentially a biologically relevant interactor following screening? (see table below)
No One Two many
Did your hunt isolate those specific partners ?
None One Two some
15.What class(es) of protein (transcription factor, phosphatase, etc) was this interactor? and where is it supposed to localize intracellularly? (please see table below)
IF you don't mind saying, what cDNAs did you isolate (name/Acc Number)? (please see table below) Were putative relevant interactors obtained as multiple independent isolates, or as a single unique cDNA?
16. Is this work published Yes No (ie, is there a reference we can check for further information)?
17. *** Did you obtain proteins you believe to be trash? ***
yes, poor me! no, thank God!
18. What criteria caused you to classify this protein as trash?
Inability to reproduce interaction upon retransformation? Biological clear irrelevance, interaction with non-specific baits (which baits?), etc... please fill in below
You see, we are much more optimistic about the number of false positives that about the number of true interactors...
19. What is your final opinion of this system? All comments welcome.
20. Do you have any objection to some or all of the information you have supplied being placed in a public database? If so, please fill in the question numbers you wish to remain completely confidential prior to your publication.
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