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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


Hello,
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
somhow.
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
NAYERS at UTMEM1.UTMEM.EDU



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