Luciferase Assay pH-dependent Fox-activity
kyle.kisor
Abstract
Working protocol for how to perform luciferase assay. Worked for Fox reporter
Before start
Read entire protocol before starting
Steps
Plate Cells
- Plate 500K cells/well for each conditon
Incubate o/n before transfection
Transfection
For each transfection condition create the following tube mixtures. ( Volumes are for 1 transfection per condition and can be scaled up)
a. Tube 1: 125uL Optimem + 4uL lipofectamine 3000
b. Tube 2: 125uL Optimem + 2uL p3000 reagent + 1 ug total DNA
flick and incubate 5 minutes
Mix together tube 1 and tube 2 and incubate 15 min
Add dropwise to cells and incubate o/n
Incubate 8 hours
Change media to fresh media or media + 10mM EIPA
48 hours incubation
Incubate cells for 48 hours after transfection. Change media at 24 hours post transfection with fresh media and fresh media + EIPA
Luciferase Assay
Slowly thaw luciferase reagent in room temp water
Wash gently each well with PBS
Add 500uL of luciferase reagent directly to plate and nutate for 10 min RT
Scrape each well with cell scraper and put into fresh microfuge tube
Spin down 13,000 rpm 5 min at RT
Add 100uL of supernatant to 4wells of white 96well plate. Wait 10 minutes
Read luciferase on plate reader
For Renilla reading, calculate reagent needed for 100uL each well using a 1:100 dilution of reagent: buffer
Add 100uL of renilla reagent to each well and wait 10 min before reading
