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beta gal substrate sensitivity in two-hybrid

nancy ayers nayers at utmem1.utmem.edu
Wed Mar 13 16:16:28 EST 1996

I am looking for any information or anyone that has used other beta gal
substrates, besides X-gal and ONPG, to assay interactions in the yeast
two-hybrid system.  My problem is this:  I am looking for interacting
regions within an oligomeric protein composed of 4 subunits/ protomer and
4 protomers/enzyme.  Therefore, I know that these subunits must interact
Yet in using the Gal4 two-hybrid system from Clonetech in their HF7c
strain I can get His reporter activity with my constructs, yet by filter
lift assay with X-gal and liquid culture assay with ONPG, I see nothing
with any transformant.  The postive control from Clonetech (p53/T-antigen
vectors) works however.  It seems strange that with a system highly prone
to false postives, I get only negatives.  My thinking is that the levels
of beta gal are so low, due to truncation of ADH promoter, that the
substrates are simply not sensitive enough. I am using the pGBT9/ pGAD424
vectors and would like NOT to have to reclone all my constructs into new
vectors. I have found a fluorometric substrate ,
methly-umbelliferyl-B-D-galactopyranoside (MUG) which is used in a lot of
mammalian cells lines with a lacZ reporter but I have not heard of anyone
using this substrate with yeast.  I know from the literature that itis
more sensitive than ONPG but I do not know about it vs. X-gal.  I was
wondering if anyone has had similar problems and/or has used this
fluorescent substrate (or another one AMPGD, which emits photons and is
highly sensitive) in yeast.  Any and all comments would be greatly
appreciated. Thank you!

Nancy Ayers

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