Luciferase Reporter Assay
| Materials | |
|---|---|
| Ice bath for buffers, Luciferin and lysates PBS
rubber policeman (cold room) and Eppis for lysates, Eppi centrifuge at RT Lysis buffer (1M DTT → 2 µl per ml) (fridge 4°C) Assay buffer (1M DTT → 10 µl per 10 ml; 200 mM ATP → 100ul per 10 ml) (fridge 4°C) Luciferin aliquots (-20°C freezer) Multipipette and plastic box for solution (cell culture room) | |
| Assay buffer (100ml): | 25 mM Tris pH 7.8 [1 M] → 2.5 ml
4 mM EGTA [100 mM] → 4 ml 20 mM MgSO4 [1 M] → 2 ml adjust pH with phosphoric acid again or KOH/NaOH to definitely get pH 7.8 shortly before use: add 10 µl = 1 mM DTT [1 M] and 100 µl = 2 mM ATP [200 mM] per 10 ml |
| Lysis buffer (100ml): | 25 mM Tris pH 7.8 [1 M] → 2.5 ml
4 mM EGTA [100 mM] → 4 ml 1 % Triton X-100 → 1 ml 10% glycerol → 10 ml shortly before use: add 2 µl/ml = 2 mM DTT [1 M] |
| D-Luciferin (110 ml): | Dissolve 0.17g DTT in 110 ml ddH2O, take 1 ml of this solution and add to 25 mg D-Luciferin, add 20 µl of 10N NaOH for dissolving Luciferin (careful: too much causes oxidation! → green solution), dilute premixture into remaining 109 ml DTT solution, aliquot and freeze at -20°C! |
Procedure
- Wash transfected cells (HEK 293) with PBS, add lysis buffer (1.3 ml per 10 cm dish), incubate at 4°C, 15 min
- scrape cells with rubber policeman, transfer to Eppi tubes, homogenization of DNA with syringe
- Centrifugation at 13 500 rpm, 5 min, RT
- Prime and install Luciferin solution at dispenser of Varioscan plate reader; submit 10-15 µl of supernatant in each well of white 96-well reader plate and measure fluorescence (mKate 588/633 nm). Add Assay buffer with multipipette (for 15 µl lysate 215 µl)
- After dispensing D-Luciferin solution (25 µl for 15 µl lysate) measure luminescence with different parameters or perform a kinetic curve (design your own protocol for your purpose)
- Data Analysis L/mKate (normalization)