Biology Reference
In-Depth Information
catalyzes the acetylation of the substrate chloramphenicol. Whereas chloram-
phenicol is soluble in aqueous solutions, acetylated forms of chloramphenicol
are lipid soluble and diffuse into the scintillation cocktail. Accumulation of
radioactivity in the water-immiscible phase is measured by liquid scintillation
counting. The samples are incubated at room temperature (or at 37
C) and
counted hourly. CAT activity is represented as the linear regression slope of
the data plotted as cpm of product vs time of incubation and is expresses as
cpm/h/
°
g protein after normalizing to the protein content of the sample assayed.
(
See
Notes 5
and
6
.)
µ
1.
Rinse cells 2X in ice-cold PBS.
2.
Scrape cells with a rubber policeman in 1 mL of CAT scraping buffer and trans-
fer to 1.5-mL tube.
3.
Recover cells by centrifugation at 12,000
g
for 15 s.
4.
Resuspend cell pellet in 100
µ
L of CAT extraction buffer.
5.
Extract proteins with three consecutive freeze/thaw cycles (freezing on dry ice
and thawing at 37
°
C for 1 min).
6.
Heat cell extracts at 65
°
C for 15 min to inactivate potential deacetylase activity.
7.
Centrifuge extract for 5 min at 12,000
g
at 4
°
C and place supernatant into a clean
1.5-mL tube.
C until assayed.
9. Label scintillation vials for each sample and include a blank and a positive control.
10. Pipet up to 50
8.
Store extract at -80
°
µ
L of the sample into a scintillation vial; adjust volume up to
50
µ
L with CAT extraction buffer. For the blank, add 50
µ
L CAT extraction
buffer. For positive control, add 49
µ
L CAT extraction buffer and 1
µ
L of puri-
fied CAT (0.1 U/
µ
L).
11.
Prepare enough CAT reaction mix for total samples to be assayed (200
µ
L for
each sample).
See
Table 1
.
12.
Add 200
µ
L of the CAT reaction mix to each sample.
13.
Gently overlay sample with 5 mL of scintillation cocktail. Do not mix.
14.
Incubate at room temperature (or 37
°
C).
15.
Count each h for 4 h. The reaction is usually linear for at least 4 h.
16.
For each sample, plot cpm versus time and calculate the linear regression line.
CAT activity (cpm/h) is normalized to protein or DNA content of the sample
(see
Note 5
).
3.3. Luciferase Reporter Gene Assay
in Transfected RCS Cells Using FuGene
The luciferase assay is more sensitive than the CAT assay. The use of two
luciferase reporter enzymes,
firefly
and
Renilla
luciferase, make it possible to
cotransfect the “experimental” reporter construct with the “control” reporter
construct as an internal control. Because the two enzymes have different sub-
strate specificity and kinetics, their activity can be measured in a single test
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