Two Hybrid False Positives?

Joe Boutell boutell at cf.ac.uk
Mon Nov 18 10:01:14 EST 1996


als152 at psu.edu wrote:
>Hello everyone,
>
>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.
>
>Andrea Skirpan
>Penn State
>als152 at psu.edu

Hiya!
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,
Cheers,
Joe Boutell.




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