Luciferase Assay Buffer: (Promega)
Stock: 9 ml
500 mM Tricine 360 µl
100 mM Mg-carbonate 97
6 ml 3 ml final conc.
240 µl
64
120 µl
32
1 M MgSO
4
50 mM EDTA
1 M DTT
10 mM coenzymeA
100 mM luciferin
100 mM ATP
DDH
2
O
24
18
299
243
42.3
47.7
7869
16
12
198
162
28.2
31.8
5248
Buffer has to be room temperature before using.
NP40 Lysis Buffer:
Stock: Volume: Final Concentration:
7.5
6
99
81
14
16.5
2624
20 mM Tricine
1.07 mM MgCO
2.67 mM MgSO
0.1 mM EDTA
33.3 mM DTT
4
3
270 µM coenzymeA
470 µM luciferin
530 µM ATP
2.
3.
4.
1 M Tris, pH 7.8 10 ml
10% NP40
DDH
2
O
5 ml
85 ml
100 mM Tris, pH 7.8
0.5% NP40
Final volume is 100 ml
Add DTT to final conc. of 50 mM right before use. 2 ml buffer + 1 µl 1 M DTT.
PROTOCOL:
1. Wash plate 3X with PBS. Make sure all liquid is sucked up.
Add 150 µl of NP40 lysis buffer to plate and incubate for 15 minutes.
Tilt plate and collect lysate. Freeze in -80 freezer if not using right away.
Turn on luminometer. Do wash program with DDH
2
O - 5X and then with luciferase assay buffer 3X. Make sure the assay buffer is room temp before use.

5. Measure 10 µl of cell lysate into tube and put in machine and measure. Do each sample in triplicate. Average triplicate. Wash luminometer after use with DDH
2
O.
6. Also measure B-gal activity to determine cell number and accuracy.
B-Gal Assay:
80 µl DDH
2
O
20 µl cell lysate
20 µl 1 M ONPG (4 mg/ml made up in DDH
2
O)
Do each sample in duplicate in ELISA plate. Incubate at 37 C for 1 hour plus.
Measure OD
420
. Stop reaction with: 100 µl of 600 mM Na
2
CO
3
. Measure OD
420 again.
Average duplicate.
Divide Luciferase number by B-gal number to get your accurate reading normalized for cell number.