regulation of arabidopsis tga transcription factors by cysteine ...

3.5 Enzymatic assays
3.5.1 β-galactosidase assay
To display β-galactosidase activity, three isolated colonies of each
transformation experiment were transferred to a fresh plate and grown overnight at
28°C. The colonies were lifted from the plate using a 0.45 micron nylon transfer
membrane (GE Osmonics, Trevose, PA), which was then submerged in liquid nitrogen
for 5-10 seconds to lyse the cells. The thawed membrane was laid on a piece of
Whatman #1 filter paper saturated with Z-buffer (100 mM Na2PO4, 10 mM KCl, 1 mM
MgSO4, 0.27% β-mercaptoethanol and 0.024 mM X-GAL), and incubated at 28°C
overnight (Kohalmi et al., 1997).
3.5.2 Quantitative β-glucuronidase (GUS) assay of yeast cells
An overnight preculture was used to inoculate a fresh culture so that the OD600
was 0.5-0.8 after 2-3 hrs incubation at 28°C. A 1.9 ml aliquot of cell suspension was
pelleted in a microcentrifuge and 200 µl of GUS extraction buffer (50 mM NaPO4, pH
7.0, 10 mM Na2EDTA, pH 8.0, 10 mM β-mercaptoethanol, 0.1% Sarcosyl, 0.1%
Triton X-100) and 100 µl volume of 425-600 micron, acid-washed glass beads (Sigma,
G-8772) was added to the pellet. The cells were lysed by seven repetitions of vortexing
for 30 sec. and cooling on ice for 30 sec., and cell debris was removed by centrifugation.
At this point the cell extract was used for GUS analysis or frozen at -80°C for future use.
To 450 µl of 1 mM methyl-umbelliferyl-β-D-glucuronide (MUG) solution, 50 µl
of cell extract was added, followed by incubation at 37°C. At various time points (0, 1,
2, and 3 hrs) 100 µl aliquots were taken and mixed with 900 µl of 0.2 M Na2CO3 stop
buffer. GUS activity was determined by measuring methyl-umbelliferone (MU)
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