beta gal substrate sensitivity in two-hybrid

Matthew Sachs msachs at ADMIN.OGI.EDU
Wed Mar 13 16:57:37 EST 1996


The Tropix Galacto-Light Plus chemiluminescent reporter assay for
beta-galactosidase has worked very well for us (for Kim Russell in James
Cregg's lab and for myself) in assays of Saccharomyces and Neurospora whole
cell extracts; it is extremely sensitive.   We use it with a scintillation
counter that allows single photon counting.

In experiments reported by VK Jain and IT Magrath (A chemiluminescent assay
for quantitation of beta-galactosidase in the femtogram range:  application
to quantitation of beta-galactosidase in lacZ-transfected cells.  Anal.
Biochem. 199: 119-124, 1991) the Tropix substrate allowed detection of 2 fg
of enzyme, MUG allowed detection of 200 fg of enzyme, and ONPG, 100 pg of
enzyme.

Tropix's address:

Tropix, Inc.
47 Wiggins Avenue
Bedford, MA  01730
Phone:  617 271-0045 or 800 542-2369
Fax:  617 275-8581

>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

-----------------------------------------------------------
Matthew Sachs
Department of Chemistry, Biochemistry and Molecular Biology
Oregon Graduate Institute of Science and Technology
20000 NW Walker Road
P.O. Box 91000
Portland, OR  97291-1000
503 690-1487 Phone
503 690-1464 Fax
msachs at admin.ogi.edu





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