96-well b-gal assays

john.jackson at PIERCENET.COM john.jackson at PIERCENET.COM
Thu Jul 22 08:32:18 EST 1999

I realize that this essentially constitutes an advertisement, but since our own
in-house advertising hasn't hit yet, I felt that I should post this message so
researchers know a little about this product and can directly ask me any
questions about it.

Pierce has introduced a Yeast b-gal Assay Kit (prod. 75768), which is based on
the Y-PERtm chemical lysis reagent.  Researchers have also experienced great
success using this kit on bacteria, such as E.coli, following the supplied
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.

1. 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.

2.  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.

3, 4:  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.  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.

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:

John C. Jackson, Ph.D
 Research Scientist
 Pierce Chemical
 3747 N. Meridian Rd.
 Rockford, IL 61101
 email: John.Jackson at mail.piercenet.com

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