>To: yeast at net.bio.net>From: "Fred Kippert" <fkippert at srv0.bio.ed.ac.uk>
>Date: Sat, 10 Oct 1998 19:37:15 +0000
>Subject: Re: 96-well Beta-gal assay for yeast ?
Bjorn Johansson <bjorn.johansson at tmb.lth.se> wrote:
> I would like to ask a question regarding microtiter plate Beta-Gal
> assays for yeast. I know this issue has been discussed here before,
> but I haven't found any concluding answer.
> I would like to grow my yeast cells in a 96-well plate, permeabilize
> them and assay for Beta-galactosidase with ONPG directly in the
> plate.
> I have read the article of F. Kippert 1995, FEMS Microbiology
> letters "A rapid permeablization procedure for accurate quantitative
> determination of Beta-Gal. in yeast". There is a method described
> using Sarcosyl and DTE and no need for vortexing. I wonder if any
> one has done this in 96-well plates ?
Pierce has introduced a Yeast b-gal Assay Kit , which is based on the Y-PERtm
chemical lysis reagent (completely unrelated to the Kippert protocol and
reagents). In comparisons, I can say that our kit works far faster and gives
much higher activity than the Kippert or any other available protocol using ONPG
and allows assays to be done in microtiter plates straight from cells in media.
The kit includes 5 protocols, which are summarized below. In all cases, 2X
assay buffer (with ONPG) is mixed 1:1 with the Y-PERtm lysis reagent, and this
mixture is then added directly to cells as they are growing in media. No
pelleting of cells, making extracts, freeze/thaw, chloroform/SDS, etc. Lysis is
immediate and complete which allows for faster assays, and the completeness of
lysis combined with the gentle nature of the lysis solution (to proteins) allows
for more sensitive assays (typically higher activity numbers as compared to
other methods, while ratios between control and test conditions remain what is
normally seen.
Protocols:
1,2: Microwell assays (ie 96 well plate). Yeast (or bacteria) growing in media
can be assayed directly by first determining OD600, then adding an equal volume
of lysis/assay buffer to the cells in media. No pelleting of cells is required.
Lysis/permeabilization is extremely rapid and complete, reducing assay time and
variability while simultaneously increasing signal compared to other protocols.
One protocol is for an unstopped assay, since plate readers can read the entire
plate at once, if you're only doing a one plate assay and reading it
immediately, there's no need to stop the assay. The other protocol is for a
stopped assay. Enough reagents for 3-5 96-well microwell plates.
3. Colony qualitative assay: an alternative to filter lift assays, allows for
the researcher to pick a small part of a colony off a plate and assay it for
b-gal activity by resuspending it in the lysis/assay buffer. "Strong"
interactions in 2-hybrid screens turn yellow in under 1 minute, a beta-testers
"very weak" interaction took 2 hours. Enough reagents for at least 100 assays.
4. Colony quantitative assay: with fresh colonies we've been able to get very
reproducible results using part of colonies picked off solid media. This time,
the colony is resuspended in lysis reagent, OD600 read, then assay initiated by
adding assay buffer. Enough reagents for 100 assays.
5: Microcentrifuge tube assay: Starting material here is a larger culture, say
5mL. Again, an aliquot of cells from the culture is added to lysis/assay buffer
in a microcentrifuge tube which initiates the assay. Cell debris can then be
easily removed by centrifugation and the supernatant transferred to a cuvette.
Enough reagents for 140 assays.
If there are any questions or comments, feel free to contact me directly. The
instruction book is available on our web site:
www.piercenet.com
John C. Jackson, Ph.D
Research Scientist
Pierce Chemical
3747 N. Meridian Rd.
Rockford, IL 61101
phone:815-968-0747
facsimile:815-968-0814
email: John.Jackson at mail.piercenet.com
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