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 ?
>> Some formulas for calculating Beta-gal activity from permeabilized
> cells involve subtracting OD550 from OD420 to make up for cells in
> the cuvette. Is it possible to leave out the centrifugation step and
> measure OD420 and OD550 on the cellsuspension and still get a good
> activity reading ? I don't have a centrifuge for microtiter plates,
> so I would prefer not to have to spin down the cells.
>
Your can use the sarcosyl permeabilization protocol in microplates.
Although no vortexing is required, you have to make sure that the
solutions are well mixed. Subtraction of the OD at 550 nm is ok, but
accuracy will not be as good as after removal of the cells. For most
applications it should be good enough, anyway better than
SDS / chloroform.
Cheers
Fred Kippert
_____________________________________
Fred Kippert
Biological Timing Lab
Institute of Cell, Animal and Population Biology
University of Edinburgh
King's Buildings
Edinburgh EH9 3JT
Scotland, UK
Tel 0131 650 5488
FAX 0131 650 6564
e-mail f.kippert at ed.ac.uk
URL: http://helios.bto.ed.ac.uk/icapb/btl/btl.html
