Two Hybrid False Positives?
boutell at cf.ac.uk
Mon Nov 18 10:01:14 EST 1996
als152 at psu.edu wrote:
>We screened a yeast two-hybrid library in an effort to identify
>proteins that interact with a our bait protein. We isolated a clone
>that was positive on the B-gal assay; however, I am not always able to
>reproduce this result. I've tried filter assays and sometimes I get a
>faint blue color, sometimes no color at all or background levels. I
>recently tried a liquid assay however this did no give a positive
>result. Data from independent method suggests that there may be a weak
>interaction. Are there any two hybrid assays (CPRG or agarose overlay
>for example) that are more sensitive. Is there any reliable way to
>destinguish between a weak interaction and a false positive? Any
>suggestions would be greatly appreciated.
>als152 at psu.edu
I've encountered a similar problem during my screens. I got a really
strong blue on filter assaying the screen (less than an hour to go blue).
However, after getting this blue out of the yeast and retransforming it
back in with my bait, it then took up to 24 hours to go blue and was
really faint. I found the way around this was to plate the
retransformants on SD-His, Trp, Leu, not just SD-Trp, Leu. It seems like
the extra His selection at this step also selects for better LacZ
reporter expression, and hey presto, they go blue in under an hour again.
Oh well, hope that helps,
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