Reporter assays
O. Gjoerup 9-14-99
1. Choose an appropriate cell line to study reporter activity in. Background should be low
and fold activation (ideally) high. Seed the cells a day in advance into p60 dishes. For
NIH 3T3 cells, transfections are done at 30-40 % confluency. However, for some cell
lines, such as SAOS-2, transfect at 70-80 % confluency (because of higher cell death
when transfecting).
2. Transfect by calcium phosphate or another transfection protocol known to work for the
particular cell line.
Use 1-2 µg of the reporter plasmid and 1-3 µg of expression plasmid (Use a lot if
expression is directed from a relatively weak promoter like SV40, and correspondingly
less when using a strong promoter such as CMV). Include also an internal control
plasmid, e.g. 0.5-1 µg pCMX beta-gal. Total amount of DNA added should be
normalized to appr. 5 µg, adding up with empty vector when appropriate.
In each assay you should have a vector control containing only the reporter plasmid plus
vector and internal control. This will reveal your basal transcriptional activity. If
possible, a dose titration of expression plasmid is recommended.
3. In some cases, serum starvation may improve on the actual fold activation (e.g. in the
case of the c-fos promoter). If you so desire, transfer the cells to low serum (0.2-0.5 %)
for the last 24 hrs before harvesting. Cells are most often harvested 48 hrs after
transfection was initiated.
4. Wash cells on the dishes 2x with PBS, then add 400 µl 1x reporter lysis buffer
(Promega, E397A) to extract for 10 min. (rock every 3 min.). Scrape with a cell scraper
and transfer into eppendorf tubes (same cell scraper can be used throughout, but dip it in
between in a large beaker with water to prevent carry-over). Vortex. Centrifuge at
10,000xg for 1 min and transfer supernatant to a new set of eppendorfs.
5. Use 20 µl for luciferase assay; the rest for beta-gal assay.
Luciferase assay: Aliquot 100 µl of luciferase assay reagent (Promega, stored in 1 ml
frozen aliquotes) into specially designed 'cuvettes' (e.g. #2050-500 from Analytical
Luminescence Laboratory).
Bring the cuvettes containing luciferase reagent, the lysates and a timer to the Berthold
LB9507 luminometer. Use a program with 15 s 'delay time' and 10 s 'integration time'
when measuring. Do it manually - add 20 µl lysate to the 100 µl luciferase assay reagent,
mix and place in luminometer. Hit start button after 15 s. The RLU (relative luciferase
units) should be printed out for each sample.
6. Beta-gal assay.
Make up a 10x beta-gal assay reagent as follows:
8.8 mg/ml ONPG (o-nitrophenyl--D-galactopyranoside)
10 mM MgCl2
100 µl 1 M stock
0.45 M beta-mercapto ethanol
88.0 mg
0.3 ml
to a total of 10 ml with PBS
Stock can be stored frozen.
Mix appr. 380 µl lysate + 40 µl 10x beta-gal assay reagent - incubate at 37 C until faint
yellow color develops, then stop with 700 µl 1M Na2CO3. Measure absorbance at 420
nm in a spectrophotometer.
Calculate (RLU/ beta-gal activity) to correct the luciferase values for transfection
efficiency.