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 ERDC TN-DOER-C30
February 2003
PLATE 1
Ponasterone A +
Ponasterone A (4 concentrations) + Environmental samples (6 samples,
Iso-octane
3 replicate extracts per sample)
0
0.1 M
0.1 M
0.1 M
0.1 M
1
1
1
0.3
10
10
10
1
100
1-0
100
3
0.1 M
0.1 M
0.1 M
10
1
1
1
30
10
10
10
100
100
100
100
11) Dose the remaining 8 environmental samples in Plate 2 similar to Plate 1,
replacing the Ponasterone dose-response series (first three columns) with
environmental samples.
12) Write down the time and date of dosing on the lids of the plates.
IV. Day 4 (ASSAY TAKEDOWN)
The exposure to the Ponasterone A standard and environmental samples is terminated at 48 hr.
The cell medium is removed, and the cells washed and lysed. The lysates are then transferred into
the 96-well plate and read. The spectrophotometer is programmed to monitor the formation of the
end product at 405-420 nm every minute for 15 min at 37 C.
A.
MATERIALS AND EQUIPMENT
Multichannel motorized pipettor (250 L)
PBS, 1x, nonsterile
o-Nitrophenyl-Galactopyranoside (ONPG) substrate (see "REAGENT PREPARATION"
section)
Buffer A (see "REAGENT PREPARATION" section)
-mercaptoethanol
Cell lysis buffer (1x), PHARMINGEN #556871 - 2 aliquots
50-mL solution basins, nonsterile
Vortex (with microplate adaptor holder)
Inverted microscope
Countertop centrifuge (with microplate adaptor holder) spectrophotometer (with
temperature control and kinetics package)
96-well plate, clear flat-bottomed, nonsterile
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