2/27/2008 RAK
Luciferase Assay Protocol
Plate cells in 6-well dishes and grow to 50-60% confluence
Transfect each well with a total of 2ug of DNA
PGL3-hERT wt (710) 1ug
Other DNAs combined 1ug
Mix DNAs together prior to transfection – enough to do each condition in duplicate.
An example experiment to look at repression and co-activation by NFX1 isoforms:
1 2 3 4
pGL3-hTERT 1 1 1 1
pZOME 1 0.5
pB-myc 0.5 0.5 0.5
N91-pZ 0.5
N123-pZ 0.5
Use 1:3 ratio of ug DNA: ul FuGENE for HFKs and 1:4 ratio for 293Ts
Make a master mix of FuGENE diluted in serum-free media (100ul per well), mix well, incubate at RT to 5 minutes (include enough for at least 2 extra transfections to allow for pipet error.)
Add diluted FuGENE to tubes of DNA dropwise (if in duplicate and using 100ul per well, add 200ul to each tube). Mix gently by tapping tube and incubate at RT for 15 minutes.
Add 100ul of transfection mix to each well dropwise and swirl to mix.
Incubate at 37ºC for 24 hours.
To harvest cells, rinse with PBS once or twice, removing as much PBS as possible.
Add 150-200ul of 1X reporter lysis buffer to each well and tap gently to mix well.
Freeze plate at -80ºC for at least 30 minutes to help with cell lysis.
Thaw plate at RT or in 37ºC incubator and scrape cells off plate.
Transfer to 1.7ml eppendorf. Spin down cell debris at 14K for 10-15 minutes of 4ºC.
Assay lysates for luciferase activity and protein concentration.
For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene Falcon Tube.
Read on luminometer in the gel room using Promega Luciferase Assay Substrate thawed and mixed at RT. Values are Relative Light Units (RLU).
Use BioRad assay for protein concentrations in 20ul of lysate. Use these values to normalize luciferase values (calculate RLU/ug protein by dividing luminometer reading by protein concentration).
Notes:
When looking at activity of the hTERT promoter, the background activity will be much higher in hTERT (+) cells such as 293T cells than in hTERT (-) cells such as HFKs. This may significantly influence the levels of repression or activation by your transfected DNAs. Also, very high luciferase activity values will not print out on the luminometer and will need to be written down (I think it’s any value over 99,999,999???)